Pacemake ion channel proteins and uses thereof

ABSTRACT

The present invention provides an isolated nucleic acid encoding a BCNG protein or a portion thereof or BCNG-related protein or a portion thereof. The present invention further provides a method for identifying a nucleic acid in a sample which encodes a BCNG protein or a BCNG-related protein. The present invention also provides a method for testing whether a compound affects the expression of a BCNG protein or a BCNG-related protein. In addition, the present invention further provides a method for identifying a compound capable of interacting with a BCNG protein or a BCNG-related protein. Also, the present invention provides a method for identifing a compound capable of modulating BCNG protein or BCNG-related protein activity. Further, the present invention also provides a method of treating a condition in a subject which comprises administering to the subject an amount of the provided compound, effective to treat the condition. Finally, the present invention provides a pharmaceutical composition which comprises the provided compound and a pharmaceutically acceptable carrier.

[0001] This application is a continuation-in-part of U.S. Ser.

[0002] No. 08/997,685, filed Dec. 23, 1997, the content of which ishereby incorporated into this application by reference.

[0003] Throughout this application, various publications are referencedby author and date. Full citations for these publications may be foundlisted alphabetically at the end of the specification immediatelypreceding the Sequence Listing and the claims. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart.

BACKGROUND OF THE INVENTION

[0004] Introduction

[0005] Ion channels are a diverse group of proteins that regulate theflow of ions across cellular membranes. In the nervous system, ionchannel activity has evolved into a rapid and accurate system forintercellular communication. The electrical excitability characteristicsof each neuron is in part determined by the set of channels itexpresses. However, cells are also able to regulate the activity ofindividual channels in response to physiological or developmentalevents, and there is growing evidence that ion channels can be the siteof integration of multiple electrical and biochemical pathways.

[0006] In vivo, ion channels appear to be multimeric proteins that arecomprised of several distinct gene families, coding for channels withdistinct structural and functional properties. Within a gene family, thepotential for heterogeneity arising from the combinatorial assembly ofdifferent pore-forming and auxiliary subunits (Greene, et al., 1995).Channel properties can be modulated by second messenger cascades and candirectly bind intracellular proteins such as kinases suggesting thatthis may be an important way to efficiently target the signaling cascadeto its effector molecule. The electrical characteristics of each neuronis, in part, determined by the set of ion channels that it expresses.However, cells are also able to regulate the activity of individualchannels in response to physiological or developmental events;pore-forming (a) subunits can interact with a variety of intracellularproteins, including auxiliary (β) subunits, cytoskeleton-associatedproteins and protein kinases (Greene, et al., 1995). In addition toauxiliary (β) subunits, pore-forming subunits can interact with avariety of intracellular proteins and second messenger moleculesthemselves including G-proteins, cytoskeleton-associated proteins andprotein-kinases (Adelman, et al., 1995).

[0007] Several classes of ion channels bind directly, and are regulatedby, second messenger molecules such as cyclic nucleotides (Zagotta, etal., 1996; Bruggemann, et al., 1993, and Hoshi, et al., 1995) or Ca⁺²(Adelman, et al., 1992; Kohler, et al., 1996). Channels with thisproperty may be key elements in the control of neuronal signaling, asthey directly couple biochemical cascades with electrical activity.Cyclic nucleotide-gated channels (CNG) play a distinct role both invisual and olfactory signal transduction; their recent identification inthe hippocampus and other regions of the brain, where cAMP and CGMP areknown to mediate different forms of synaptic plasticity (Krapivinisky,et al., 1995; Frey, et al., 1993; Bolshakov, et al., 1997; and Arancio,et al., 1995), suggests that CNG-channels may also contribute to theregulation of excitability in central neurons (Kingston, et al., 1996and Bradley, et al., 1997).

[0008] The first structural gene for a K⁺ channel to be isolated was thegene encoded by the Shaker (Sh) locus in Drosophila melanogaster(Strong, et al., 1993; Papazian, et al., 1987). Its sequence is theprototype of a large and still expanding family of related genes (Kamb,et al., 1987; Warmke, et al., 1994). The properties of a number of wellcharacterized K⁺ currents, that still await a molecular definition,predicts that other members of this family are yet to be identified(Atkinson, et al., 1991).

[0009] Although the initial members of the K⁺ channel superfamily werecloned by chromosomal localization of alleles responsible for functionaldefects (Sh, eag and slo from Drosophila; (Papazian, et al., 1987; Kamb,et al., 1987; Warmke, et al., 1991; Atkinson, et al., 1991) or followingthe purification of a relatively abundant protein such as thecGMP-channel from bovine retina (Liman, et al., 1994), the most widelyused strategy for cloning new members of the K⁺ channel superfamily isby homology to these sequences. Unfortunately, this approach is not wellsuited for identifying more divergent sequences and potentially newbranches in the phylogenetic tree of the K⁺ channel superfamily.Expression cloning in Xenopus oocytes can circumvent this problem; thisimplies a pre-existing or readily detectable physiologicalcharacterization of the channel.

SUMMARY OF THE INVENTION

[0010] The present invention provides an isolated nucleic acid encodinga BCNG protein or a portion thereof. The present invention furtherprovides an isolated nucleic acid encoding a BCNG-related protein or aportion thereof. Further, the present invention provides a vector, whichcomprises cDNA encoding mBCNG-1 (ATCC Accession No. ______). Inaddition, the present invention further provides a vector, whichcomprises cDNA encoding hBCNG-1 (ATCC Accession No. ______). The presentinvention also provides an isolated BCNG protein. Further, the presentinvention also provides an isolated BCNG-related protein.

[0011] The present invention additionally provides a compositioncomprising a nucleic acid encoding a BCNG protein or a portion thereof,or a BCNG-related protein or a portion thereof and a carrier. Inaddition, the present invention further provides a compositioncomprising a BCNG protein or a portion thereof, or a BCNG-relatedprotein or portion thereof and a carrier.

[0012] Additionally, the present invention provides a nucleic acid probecapable of specifically hybridizing with a nucleic acid encoding a BCNGprotein or BCNG-related protein.

[0013] The present invention provides a method for identifying a nucleicacid in a sample which encodes a BCNG protein or a BCNG-related proteinwhich comprises: (a) contacting the sample with a nucleic acid probecapable of specifically hybridizing with nucleic acid encoding a BCNGprotein or a BCNG-related protein under conditions permissive to theformation of a complex between the nucleic acid probe and the nucleicacid encoding the BCNG protein or the BCNG-related protein in thesample; (b) determining the amount of complex formed in step (a); and(c) comparing the amount of complex determined in step (b) with theamount of complex formed using an arbitrary sequence, a greater amountof complex formed with the BCNG-specific probe indicating the presenceof a nucleic acid encoding a BCNG protein or a BCNG-related protein inthe sample.

[0014] Further, the present invention provides a method for testingwhether a compound affects the expression of a BCNG protein or aBCNG-related protein which comprises: (a) contacting a sample whichexpresses a BCNG protein or a BCNG-related protein with a compound; (b)determining the amount of expression of BCNG protein or BCNG-relatedprotein in the sample; and (c) comparing the amount of BCNG protein orBCNG-related protein expression determined in step (b) with the amountdetermined in the absence of the compound.

[0015] In addition, the present invention further provides a method foridentifying a compound capable of interacting with a BCNG protein or aBCNG-related protein which comprises: (a) contacting a sample whichexpresses a BCNG protein or a BCNG-related protein with a compound underconditions permissive to formation of a complex between the compound andthe BCNG protein or the BCNG-related protein; (b) determining the amountof complex formed between the compound and the BCNG protein or theBCNG-related protein; (c) comparing the amount of complex formed in step(b) with the amount formed in the absence of the compound, a greateramount of complex formed in the presence of the compound indicating thepresence of a compound capable of interacting with a BCNG protein or aBCNG-related protein.

[0016] Also, the present invention provides a method for identifing acompound capable of modulating BCNG protein or BCNG-related proteinactivity which comprises: (a) contacting a sample which expresses a BCNGprotein or a BCNG-related protein with a compound; (b) determining theamount of activity of the BCNG protein or BCNG-related protein in thesample; and (c) comparing the amount of activity of the BCNG protein orthe BCNG-related protein determined in step (b) with the amountdetermined in the absence of the compound, an increase or decrease inactivity indicating the presence of a compound capable of modulating theactivity of the BCNG protein or the BCNG-related protein.

[0017] Further, the present invention also provides a method of treatinga condition in a subject which comprises administering to the subject anamount of the provided compound, effective to treat the condition.

[0018] Finally the present invention provides a pharmaceuticalcomposition which comprises the provided compound and a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

[0019] FIGS. 1A-1D. Primary structure of mBCNG-1. FIG. 1A. Deduced aminoacid sequence (Seq.ID.No.:2) encoded by the mBCNG-1 cDNA. The sevenhydrophobic domains, homologous to the six transmembrane domains (S1-S6)and the pore (P) of K⁺ channels, are indicated (______). The putativecyclic-nucleotide binding site (CNBs) is marked by an (- - - ),C-terminal prolines ( . . . ) the consensus N-glycosylation site withpresumptive extracellular localization (*) are also marked. FIG. 1B.Kyte and Doolittle hydropathy plot of the predicted amino acid sequenceof mBCNG-1. The profile was generated by the Kyte and Doolittle methodwith a window size of 7 amino acids. The numbers on the top lineindicate the position in the mBCNG-1 sequence. Hydrophobic regionscorresponding to S1 through S6 and the P region lie below the zero linewhile the N-glycosylation site (*) is in a hydrophilic region between S5and P. Numbering (top line) indicate position in the mBCNG-1 sequence.Profile generated with a window size of 7 residues. FIG. 1C. Multiplealignment of the putative P region of mBCNG-1 with the P regions ofDrosophila Eag (DEAG), mouse Eag. (MEAG), human Erg (HERG), α-subunit ofbovine retinal CNG-channel (BRET-1), and β-subunit of human retinalCNG-channel (HRET-2). Arrowheads mark the residues 344 and 352 (seeExample 1). FIG. 1D. Alignment of the (CNBs) of BCNG-1 with thecorresponding site in the rat olfactory CNG-channel (ROLF-1), bovinecGMP-dependent protein kinase (PKG), bovine cAMP-dependent proteinkinase (PKA), and catabolite activator protein of E. coli (CAP).Continuous lines mark α-helical (α) and β-strand (β) elements of thesecondary structure elements of CAP, while asterisks indicate specificamino acids that appear to lie close to the cAMP molecule in the CAPcrystal structure.

[0020] FIGS. 2A-2D. mBCNG-1 is a 132 kDa glycosylated protein. FIG. 2A.Western blot analysis of BCNG-1 protein in a mouse brain extract. Ten μgof a total brain SDS-extract was loaded per strip then probed with αq1(1) or αq2 (3) antiserum or, strip 2 with αq1 (2) or αq2 (4) antiserumpreadsorbed with the GST-d5 fusion protein. The arrow marks the positionof the specific signal, of corresponding to the native mBCNG-1 protein.FIG. 2B. Western blot using the αq1 antiserum against: total brainextract (1), total brain extract pre-treated with N-glycosidase F (2)and in vitro translated mBCNG-1 protein (3). Positions of molecularweight standards are shown on the left. Also shown, Western blotcontaining 10 μg of proteins from each of the indicated brain tissueswhich was tested with antisera against mBCNG-1 and showing widespreadexpression of the mBCNG-1 protein in mouse brain. FIG. 2C. indicatesreactivity with αq1. FIG. 2D. indicates reactivity with αq2.

[0021]FIG. 3. Northern blot analysis of mBCNG-1 expression in differentmouse tissues. Two μg of poly(A)⁺ RNA from each of each of the followingtissues was used: heart (H), brain (B), spleen (S), lung (Lu), liver(Li), skeletal muscle (M), kidney (K) and testis (T) were loaded. Thefilter was probed with a DNA fragment encoding amino acids 6-131 of themBCNG-1 sequence. A probe corresponding to amino acids 594-720recognized the same bands, confirming that the cDNA fragments isolatedfrom the γgt10 and pJG4-5 libraries are from a contiguous mRNA sequence.Positions of molecular weight standards are shown on the left.

[0022]FIG. 4. In situ hybridization analysis of mBCNG-1 expression inthe brain. Parasagittal section of a mouse brain probed with anantisense oligonucleotide directed to the mRNA region corresponding toamino acids 648-657 of the mBCNG-1 sequence. Abbreviations: nCtx,neocortex; Hp, hippocampus; Crb, cerebellum; BrSt, brainstem.

[0023] FIGS. 5A-5F: Immunohistochemical analysis of mBCNG-1 expressionin the brain. Parasagittal sections of a mouse brain were stained withαq1 and α2 antisera. The patterns of mBCNG-1 expression detected withthe two different antisera were identical, and in both cases thestaining was entirely abolished by preadsorbing the sera with the GST-d5fusion protein. mBCNG-1 immunoreactivity in the cerebral cortex. FIGS.5C-5D. mBCNG-1 immunoreactivity in the hippocampus. In FIG. 5C, thearrow shows the position of the hippocampal fissure; areas CA₁, CA₃ anddentate gyrus (DG) are labeled. FIG. 5D shows a detail of the stratumpyramidale of area CA₃. FIGS. 5E-5F. mBCNG-1 immunoreactivity in thecerebellum. (FIG. 5A, FIG. 5C, FIG. 5E: 60×; FIG. 5B, FIG. 5D, FIG. 5F:100×) magnification).

[0024] FIGS. 6A-6B. Southern blot analysis of mouse genomic DNA. 4 μg ofmouse genomic DNA were loaded onto each lane following digested with EcoRI (1), Hind III (2), Bam HI (3), Pst I (4) or Bgl II (5). The filterwas probed with a DNA fragment encoding amino acids 269-462 of theBCNG-1 sequence at high (FIG. 6A) and (FIG. 6B) low stringency.Positions of molecular weight standards are shown on the left.

[0025] FIGS. 7A-7C. Schematic representation of the mouse and human BCNGclones. FIGS. 7A-7B. Predicted structure of mBCNG-1 (Santoro et al.,1997) with six transmembrane domains (S1-S6), pore region (P), cyclicnucleotide binding site (CNBs) and long C-terminal tail, including apolyglutamine stretch (Q). The predicted sequences encoded by thepartial cDNA clones of three other mouse and two human BCNG genes areshown in a tentative alignment to mBCNG-1. Lines with double-headedarrows above the sequences indicate if the fragment was obtained from acDNA library (γgt10 or pJG4-5), RT-PCR reaction, or EST database.

[0026] Dashed lines with double-headed arrows underneath the sequencesindicate the position of probes used herein (see Examples 1-5 inExperimental Details section). Hashed box in the 5′ region of mBCNG-4indicates the position of the probable intron in the M28-EST clone. FIG.7C. Percent sequence similarity among the mouse and human BCNG genes.The alignments were performed by comparing only the core region of theproteins, corresponding to amino acids 111-419 (numbering according tomBCNG-1, see FIG. 8), and including transmembrane domains S1-S6. ThemBCNG-4 sequence was not included in this alignment. However, limitedalignment within the available cyclic nucleotide binding domain sequenceof mBCNG-4 (aa 529-592, numbering according to mBCNG-1, see FIG. 8)shows a 79% similarity to mBCNG-1.

[0027] FIGS. 8A-BB. Mouse and human BCNG protein alignments. Tentativealignment of the predicted amino acid sequences for the four mouse(mBCNG-1, 2, 3 and 4) and two human genes (hBCNG-1 and 2). The proposedstructural features of the protein (putative transmembrane regions, poreregion and cyclic nucleotide binding site) are indicated (see also FIG.0.5). (−) indicates residues identical to mBCNG-1; divergent residuesare otherwise reported. (.) indicates a gap (or deletion) in the alignedsequences. (*) at end of sequence indicates stop codon. (*) aboveposition 327 marks N-glycosylation site of mBCNG-1. The arrow marks thesingle consensus PKA phosphorylation site present in BCNG-1 and BCNG-2.

[0028] FIGS. 9A-9D. Northern Blot Analysis of Mouse BCNG GeneExpression. Multiple Tissue Northern blot, containg 2 pg of polyA+ RNAfrom each of the following mouse tissues: heart (He), brain (Br), spleen(Sp), lung (Lu) liver (Li), skeletal muscle (Mu), kidney (Ki) and testis(Te), was hybridized to DNA fragments corresponding to the indicatedBCNG genes. Molecular size markers are indicated on the left.

[0029] FIGS. 10A-D. Northern Blot Analysis of Human BCNG GeneExpression. FIGS. 10A-10B. Multiple human tissue Northern blot,containing 2 mg of polyA+ RNA from each of the following tissues: heart(He), brain (Br), placenta (Pl), lung (Lu) liver (Li), skeletal muscle(Mu), kidney (Ki) and pancreas (Pa), was hybridized to DNA fragmentscorresponding to the indicated BCNG genes. FIGS. 10C-10D. The samefragments were used to probe a human Brain Multiple Tissue blot,containg 2 pg of polyA+ RNA from each of the following tissues: amigdala(Am), caudate nucleus (Cn), corpus callosum (CC), hippocampus (Hi),total brain (Br), substantia nigra (SN), subthalamic nucleus (Sn) andthalamus (Th). Molecular size markers are indicated on the left.

[0030]FIG. 11. Schematic representation of the general architecture ofthe BCNG channel proteins based on homology to the voltage-gated K⁺channels and the cyclic nucleotide-gated channels.

[0031]FIG. 12. Alignment of the S4 voltage sensing regions of theprototypical voltage-gated K⁺ channel shaker and cyclic nucleotide-gatedchannel bRET1 with the S4 sequence of mBCNG-1 (Seq.ID.No.:2). Boxedresidues are positively charged amino acids present in one or more ofthe S4 sequences. The stars indicate the position of amino acids withnegatively charged acidic side chains that are present in the bRET1sequence.

[0032] FIGS. 13A-13B. FIG. 13A. Sequence alignment of functional cyclicnucleotide binding sites from catabolite activating protein (CAP, Aibaet al., 1982; Cossart & Gicquel, 1982), A and B sites of recombinantbovine R1α (PKAa and PKAb, Titani et al., 1984), bovine retinal channelα subunit (bRET1, Kaupp et al., 1989) and the catfish olfactory αsubunit (fOLF1, Goulding et al., 1992) along with the putative cyclicnucleotide binding sites of drosophila Ether-a-gogo (dEAG, Warmke etal., 1991), Arabidopsis Thaliana K transport protein (KAT1, Anderson etal., 1992) and mBCNG-1 (Seq.ID.No.:2) (described herein) The sixresidues that are totally conserved across all of the binding siteswhose functional competence has been unequivocally confirmed are markedby asterisks. The conserved arginine that forms an ionic bond with thecyclic nucleotide is indicated by an arrow labeled R559. The residue inthe third (C) α-helix that has been shown to influence coupling ofactivation to cAMP versus cGMP binding is indicated by an arrow labeledD604 (the cGMP selective substitution in bRET1). FIG. 13B. Schematicrepresentation of the cyclic nucleotide binding site of bRET1 showingthe critical interactions between the binding site and the cyclicnucleotide. This model of the binding pocket is based on the crystalstructure of CAP and bovine Rla. The cGMP is shown bound in an extendedor anti form with the cyclized phosphate making an ionic bond withArginine559 (bRET1 numbering) and the purine ring forming favorablecontacts with D604 in this cGMP selective channel.

[0033]FIG. 14. Alignment of the P loop pore forming regions of theprototypical voltage-gated K⁺ channel shaker and cyclic nucleotide-gatedchannel bRET1 with the S4 sequence of mBCNG1. The aligned channels aremBCNG-1 (Seq.ID.No.:2), Shaker (SHAK, Papazian et. al., 1987; Kamb etal., 1988), SHAW, Wei, et al., 1990) calcium activated K channel (MSLO,Pallanck and Ganetzky, 1994) the plant inward rectifier (AKT Sentenac etal., 1992), drosophila and mouse ether-a-gogo's (DEAG, Warmke et al.,1991; MEAG, Warmke and Ganetzky, 1994) Human ether-a-gogo related gene(HERG, Warmke and Ganetzky, 1994), bovine retinal α-subunit (bRET1,Kaupp et al., 1989) and human retinal β subunit (HRET-2, Chen et al.,1993). The arrows mark positions where the BCNG channels show pronouncedand potentially important changes in sequence from the normal motif seenin K selective channels.

[0034] FIGS. 15A-15B. Schematic representation of repetitive firing of apacemaker neuron and its involvement in the generation and regulation ofrhythmic firing patterns. FIG. 15A. Shows that Ih activation uponhyperpolarization following an action potential. As this is anon-selective cationic current, it carries an inward current at thesepotentials which leads to the depolarization of the cell back towardsthe threshold for firing of the next action potential. FIG. 15B. Showsthe action of sympathetic and vagal stimulation on the activity ofcardiocytes from the sinoatrial node—the pacemaker area of the heart.Norepinephrine (NE) leads to a shift in the activation of Ih towardsmore depolarized potentials which accelerates the return to the actionpotential firing threshold, and hence, leads to an acceleration of thefiring rate. In contrast, acetylcholine (ACh) shifts the activation ofIh to more hyperpolarized potentials. Thus, the current will turn onlater during the repolarization phase delaying the return tothreshold—the firing rate of the cell will thus be slowed. These changesin the activation properties of Ih are thought to be due to changes inthe concentration of cAMP with ACh lowering the concentration and NEincreasing the concentration which has been shown to alter theactivation properties of Ih. (“Principles of Neural Science” by Kandel,Schwartz and Jessell 1991).

[0035] FIGS. 16A-16F. mBCNG-1 expression gives rise to ahyperpolarization-activated current that resembles the native neuronalpacemaker current. FIG. 16A. Currents elicited by 3 s hyperpolarizationsfrom a holding potential of −40 mV to potentials ranging from −60 to−130 mV in 5 mV increments. FIG. 16B. Relation between steady-statecurrent at end of hyperpolarizing step and patch voltage. FIG. 16C. Tailcurrents recorded upon return to −40 mV following hyperpolarizations tovarious test voltages. Records shown on an expanded time scale toemphasize tail currents. FIG. 16D. Mean relation between tail currentamplitude and voltage during hyperpolarizing step. For each patch, tailcurrent data were normalized to the maximal tail current amplitudeobtained from a fit of the Boltzmann equation (see Example 4,Experimental Details section). Normalized tail currents were thenaveraged for 5 patches. Mean V1/2=−100.0 mV, slope=5.8 mV. FIG. 16E.

[0036] Activation time course of mBCNG-1 currents. Data were sampled at5 kHz and the currents during voltage steps between −105 to −130 mV werefitted by single exponential functions (smooth lines), after allowingfor an initial lag. FIG. 16F. Relationship between mean time constantsof activation and voltage. In the above experiments, the extracellularsolution was KCl/NaCl—CaCl₂, and the intracellular solution wasKCl/NaCl-EGTA (See, Example 4, Experimental Procedures).

[0037] FIGS. 17A-17C. mBCNG-1 is a cation channel that selects weaklyfor potassium over sodium. FIG. 17A. Tail currents obtained upondepolarizing steps to various test potentials following a 0.3 sec stepto −130 mV to activate the mBCNG-1 current. Test potentials ranged from−60 to +20 mV in 5 mV increments (indicated next to alternate currenttraces) The extracellular solution was NaCl-EGTA while the intracellularsolution was Kcl-EGTA. FIG. 17B. Similar tail current protocol used tomeasure reversal potential after switching to the low Cl,KAspartate-EGTA solution in the bath. FIG. 17C. Tail current amplitudeas a function of membrane voltage during tail. Open symbols representthe current amplitudes determined with the KCl-EGTA solution in the bath(o, initial measurement, E_(rev)=−32.8±2.5, n=3; □, following washout ofthe KAspartate-EGTA solution, E_(rev)=−31.2±1.6, n=3). The filledcircles represent the measurements made in the presence of theK-Aspartate solution (E_(rev)=28.2±1.6, n=4). In all three panels, thezero current level is indicated by a horizontal dashed line.

[0038] FIGS. 18A-18C. The mBCNG-1 channel is blocked by external Cs butnot by external Ba, similar to native pacemaker channels. mBCNG-1current records from an outside-out patch. In FIGS. 18A-18C, the currentat the holding potential (−40 mV) and in response to a hyperpolarizationto −130 mV are superimposed. FIGS. 18A and 18B. mBCNG-1 current recordsfrom an outside-out patch. Currents at the holding potential (−40 mV)and in response to a step to −130 mV are superimposed. The sequentialrecords were obtained when the extracellular surface was exposed to theKCl/NaCl—CaCl₂ solution (control), or solutions in which 2 mM CsCliso-osmotically replaced NaCl or 1 mM BaCl2 replaced the CaCl₂. Theintracellular solution was Kcl/NaCl-EGTA. FIG. 18C. Dose responserelationship for the inhibition of mBCNG-1 current by Cs. Data are themean from 6 patches (not all determined at each concentration). Thesolid line shows a fit of the Hill equation,I/Imax=1/{1+(IC₅₀/[Cs])^(n)}, where [Cs] is the Cs concentration, 1C₅₀is the half maximal inhibitory concentration of Cs, n is the Hillcoefficient, I is the current in the presence of Cs and Imax is theuninhibited current. The fit yields an IC₅₀ of 201 μM and a Hillcoefficient of 1.08.

[0039] FIGS. 19A-19C. The mBCNG-1 channel is directly regulated bycytoplasmic cAMP. FIG. 19A. mBCNG-1 current record from an inside-outpatch in response to hyperpolarization to −100 mV in the absence orpresence of 1 μM cAMP in the intracellular solution (KCl-EGTA). Theextracellular solution was Kcl-CaCl₂. FIG. 19B. Records from anotherpatch in the absence and presence of 30 μM cAMP (same solutions as inA). FIG. 19C. (Left panel) Tail current activation curves in the absenceand presence of 1 μM cAMP for the patch shown in panel A. Data wereanalyzed and plotted as described in FIG. 1D. Curves fit by Boltzmannrelation with following parameters: 0 μM cAMP: V_(1/2)=−100 mV,slope=5.4 mV and I_(tail, max)=−33.8 pA; 1 μM cAMP: V_(1/2)=−98 mV,slope=5.3 and I_(tail, max)=−33.7 pA. (Right panel) Mean tail currentactivation curves for patches in absence (solid circles) and presence(open circles) of cAMP. Data averaged from 5 patches. cAMPconcentrations range from 1-3000 μM, for cAMP, V_(1/2)=−98.3 mV, slope=6mV.

[0040] FIGS. 20A-C. mBCNG-2 is expressed in the sinoatrial node of theheart. FIG. 20A. PolyA+ RNA samples from ventricle, atrial andsino-atrial node of the rabbit heart were tested by RT-PCR for thepresence of mBCNG-2 or 3 transcripts. Lanes: 1) molecular weight marker;2) reaction performed in the absence of reverse transcriptase; 3)ventricle RNA; 4) atrial RNA; 5) sino-atrial node RNA; 6) PCR reactionon plasmid containing mBCNG-1 cDNA; 7) PCR reaction on plasmidcontaining mBCNG-2 cDNA; 8) PCR reaction on plasmid containing mBCNG-3cDNA. Molecular size markers are indicated on the left. The arrow on theright indicates the expected 340 bp amplification product. FIG. 20B.Southern blot analysis of the gel shown in A using a probe to mBCNG-2.FIG. 20C. Southern blot analysis of the gel in A using a probe tomBCNG-3.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention provides an isolated nucleic acid encodinga BCNG protein or a portion thereof. The present invention furtherprovides an isolated nucleic acid encoding a BCNG-related protein or aportion thereof.

[0042] In an embodiment of this invention, the BCNG protein is encodedby the sequence shown in mBCNG-1 (ATCC Accession No. ______)(Seq.ID.No.:1), mBCNG-2 (Seq.ID.No.:5), mBCNG-3 (Seq.ID.No.:9), mBCNG-4(Seq.ID.No.:11) hBCNG-1 (ATCC Accession No. ______) (Seq.ID.No.:3) orhBCNG-2 (Seq.ID.No.:7). According to an embodiment of this invention,the nucleic acid is DNA or RNA. In an embodiment of the presentinvention, the nucleic acid is cDNA. According to an embodiment of thisinvention, the cDNA has the nucleotide sequence shown in SEQ. ID. No.: 1for mBCNG-1 (ATCC Accession No. ______), SEQ. ID. No.: 3 for hBCNG-1,SEQ. ID. No.: 5 for mBCNG-2, SEQ. ID. No.: 7 for hBCNG-2, SEQ. ID. No.:9 for mBCNG-3, or SEQ. ID. No.:11 for mBCNG-4. An embodiment of thepresent invention is a vector comprising the nucleic-acid. According toan embodiment of this invention, the vector comprises viral or plasmidDNA. An embodiment of this invention comprises the nucleic acid andregulatory elements. One embodiment of this invention is a host vectorsystem which comprises the provided expression vector in a suitablehost.

[0043] Further, the present invention provides a vector, which comprisescDNA encoding mBCNG-1 (ATCC Accession No. ______). In addition, thepresent invention further provides a vector, which comprises cDNAencoding hBCNG-1 (ATCC Accession No. ______).

[0044] In an embodiment of this invention, the suitable host is abacterial cell, a eukaryotic cell, a mammalian cell or an insect cell.

[0045] The present invention also provides an isolated BCNG protein.Further, the present invention also provides an isolated BCNG-relatedprotein.

[0046] In one embodiment of this invention, the BCNG protein has theamino acid sequence shown in Seq.ID.No.:2 for mBCNG-1 (FIGS. 8A-8B),Seq.ID.No.:6 for mBCNG-2 (FIGS. 8A-8B), Seq.ID.No.:10 for mBCNG-3 (FIGS.8A-8B), Seq.ID.No.:2 for mBCNG-4 (FIGS. 8A-8B), Seq.ID.No.:4 for hBCNG-1(FIGS. 8A-8B), or Seq.ID.No.:8 for hBCNG-2 (FIGS. 8A-8B). According toanother embodiment of this invention, the BCNG-related protein has anamino acid sequence with substantial homology to the amino acid sequenceshown in Seq.ID.No.:2 mBCNG-1 (FIGS. 8A-8B), Seq.ID.No.:6 for mBCNG-2(FIGS. 8A-BB), Seq.ID.No.:10 for mBCNG-3 (FIGS. 8A-BB), Seq.ID.No.12 formBCNG-4 (FIGS. 8A-8B), Seq.ID.No.:4 for hBCNG-1 (FIGS. 8A-8B), orSeq.ID.No.:8 for hBCNG-2 (FIGS. 8A-8B).

[0047] The present invention additionally provides a compositioncomprising a nucleic acid, encoding a BCNG protein or a portion thereofor a BCNG-related protein or a portion thereof and a carrier. In anembodiment of the present invention the nucleic acid comprisessubstantially the same coding sequence as the coding sequence shown inSEQ. ID. No.: 1 for mBCNG-1, SEQ. ID. No.: 3 for hBCNG-1, SEQ. ID.

[0048] No.: 5 for mBCNG-2, SEQ. ID. No.: 7 for hBCNG-2, SEQ. ID.

[0049] No.: 9 for mBCNG-3, SEQ. ID. No.:11 for mBCNG-4 or a portion ofsuch coding sequence.

[0050] In addition, the present invention further provides a compositioncomprising a BCNG protein or portion thereof or a BCNG-related proteinor portion thereof and a carrier.

[0051] In an embodiment of this invention the BCNG protein comprises theamino acid sequence shown in Seq.ID.No.:2 mBCNG-1 (FIGS. 8A-8B),Seq.ID.No.:6 for mBCNG-2 (FIGS. 8A-8B), Seq.ID.No.:10 for mBCNG-3 (FIGS.8A-8B), Seq.ID.No.12 for mBCNG-4 (FIGS. 8A-8B), Seq.ID.No.:4 for hBCNG-1(FIGS. 8A-8B), Seq.ID.No.:8 for hBCNG-2 (FIGS. 8A-8B).

[0052] Additionally, the present invention provides a nucleic acid probecapable of specifically hybridizing with a nucleic acid encoding a BCNGprotein or BCNG-related protein. One embodiment of this invention is anucleic acid probe capable of specifically hybridizing with the providednucleic acid. According to an embodiment of this invention the probe iscapable of specifically hybridizing with the nucleic acid sequence shownin Seq.ID.No:13, Seq.ID.No:1.4, Seq.ID.No:15, Seq.ID.No:16,Seq.ID.No:17, Seq.ID.No:18, Seq.ID.No:19, Seq.ID.No:20, Seq.ID.No:21,Seq.ID.No:21, Seq.ID.No:22, Seq.ID.No:23, Seq.ID.No:24, Seq. ID.No:25,Seq.ID.No:26, Seq.ID.No:27, Seq.ID.No:28, Seq.ID.No:29, Seq.ID.No:30,Seq.ID.No:31, Seq.ID.No:32, Seq.ID.No:33, or Seq.ID.No:34.

[0053] The present invention provides a method for identifying a nucleicacid in a sample which encodes a BCNG protein or a BCNG-related proteinwhich comprises: (a) contacting the sample with a nucleic acid probecapable of specifically hybridizing with nucleic acid encoding a BCNGprotein or a BCNG-related protein under conditions permissive to theformation of a complex between the nucleic acid probe and the nucleicacid encoding the BCNG protein or the BCNG-related protein in thesample; (b) determining the amount of complex formed in step (a); and(c) comparing the amount of complex determined in step (b) with theamount of complex formed using an arbitrary sequence, a greater amountof complex formed with the BCNG-specific probe indicating the presenceof a nucleic acid encoding a BCNG protein or a BCNG-related protein inthe sample.

[0054] In one embodiment of this invention, step (a) further comprisesamplifying the nucleic acid molecule encoding the BCNG protein or theBCNG-related protein. According to an embodiment of this invention, theamplification comprises contacting the nucleic acid molecule from thesample with at least one amplification primer capable of specificallyhybridizing to mBCNG-1 (Seq.ID.No.:1), mBCNG-2 (Seq.ID.No.:5), mBCNG-3(Seq.ID.No.:9), mBCNG-4 (Seq.ID.No.:11), hBCNG-1 (Seq.ID.No.:3) orhBCNG-2 (Seq.ID.No.:7) under conditions suitable for polymerase chainreaction. In an embodiment of this invention, the amplified nucleic acidmolecule encoding the BCNG protein or the BCNG-related protein isdetected by size fractionation. One embodiment of this invention furthercomprises isolating the complex by size fractionation. According to anembodiment of this invention, the nucleic acid probe is labeled with adetectable marker. In an embodiment of this invention, the detectablemarker is a radiolabeled molecule, a fluorescent molecule, an enzyme, aligand, or a magnetic bead. According to an embodiment of thisinvention, the probe comprises the nucleotide sequence shown inSeq.ID.No:13, Seq.ID.No:14, Seq.ID.No:15, Seq.ID.No:16, Seq.ID.No:17,Seq.ID.No:18, Seq.ID.No:19, Seq.ID.No:20, Seq.ID.No:21, Seq.ID.No:21,Seq.ID.No:22, Seq.ID.No:23, Seq.ID.No:24, Seq.ID.No:25, Seq.ID.No:26,Seq.ID.No:27, Seq.ID.No:28, Seq.ID.No:29, Seq.ID.No:30, Seq.ID.No:31,Seq.ID.No:32, Seq.ID.No:33, or Seq.ID.No:34. In an embodiment of thepresent invention, the nucleic acid probe is capable of specificallyhybridizing to nucleic acid encoding mBCNG-1 (Seq.ID.No.:1), mBCNG-2(Seq.ID.No.:5), mBCNG-3 (Seq.ID.No.:9), mBCNG-4 (Seq.ID.No.:11), hBCNG-1(Seq.ID.No.:3) or hBCNG-2 (Seq.ID.No.:7). The present invention alsoprovides an isolated nucleic acid, previously unknown, identified by theprovided amplification method.

[0055] In one embodiment, the sample comprises cells or cell extract orcell lysate or a tissue sample or a biological fluid sample. Further,the present invention provides a method for testing whether a compoundmodulates the expression of a BCNG protein or a BCNG-related proteinwhich comprises: (a) contacting a sample which expresses a BCNG proteinor a BCNG-related protein with a compound; (b) determining the amount ofexpression of BCNG protein or BCNG-related protein in the sample; and(c) comparing the amount of BCNG protein or BCNG-related proteinexpression determined in step (b) with the amount determined in theabsence of the compound thereby determining whether the compoundmodulates BCNG protein expression or BCNG-related protein expression.The present invention provides such a method for screening a largenumber of compounds to determine whether one or more of the compoundsmodulates the activity of a BCNG protein or a BCNG-related protein ormodulates the expression of the nucleic acid encoding either the BCNGprotein or the BCNG-related protein.

[0056] In addition, the present invention further provides a method foridentifying a compound capable of interacting with a BCNG protein or aBCNG-related protein which comprises: (a) contacting a sample whichexpresses a BCNG protein or a BCNG-related protein with a compound underconditions permissive to formation of a complex between the compound andthe BCNG protein or the BCNG-related protein; (b) determining the amountof complex formed between the compound and the BCNG protein or theBCNG-related protein; (c) comparing the amount of complex formed in step(b) with the amount formed in the absence of the compound, a greateramount of complex formed in the presence of the compound indicating thepresence of a compound capable of interacting with a BCNG protein or aBCNG-related protein.

[0057] Also, the present invention provides a method for identifing acompound capable of modulating BCNG protein or BCNG-related proteinactivity which comprises: (a) contacting a sample which expresses a BCNGprotein or a BCNG-related protein with a compound; (b) determining theamount of activity of the BCNG protein or BCNG-related protein in thesample; and (c) comparing the amount of activity of the BCNG protein orthe BCNG-related protein determined in step (b) with the amountdetermined in the absence of the compound, an increase or decrease inactivity indicating the presence of a compound capable of modulating theactivity of the BCNG protein or the BCNG-related protein.

[0058] The present invention also provides for compounds or compositionswhich are identified through the compound screening methods describedherein, as capable of modulating the activity or expression of BCNGprotein or BCNG related protein.

[0059] An embodiment of this invention is step (a) comprising firstintroducing the nucleic acid encoding a BCNG protein or a BCNG-relatedprotein into an expression system and causing the expression system toexpress the nucleic acid under conditions whereby a BCNG protein or aBCNG-related protein is produced. Another embodiment of this inventionis wherein step (b) comprises measuring the channel electrical currentor intracellular calcium level in the presence of the compound. In yetanother embodiment of this invention, the expression system comprises acultured host cell.

[0060] In an embodiment of this invention, the BCNG protein comprisesthe amino acid sequence of mBCNG-1 (Seq.ID.No.:2), mBCNG-2(Seq.ID.No.:6), mBCNG-3 (Seq.ID.No.:10), mBCNG-4 (Seq.ID.No.:12),hBCNG-1 (Seq.ID.No.:4) or hBCNG-2 (Seq.ID.No.:8) or a portion thereof.In one embodiment of the present invention, the sample comprises a cell,cell lysate or cell-free translation. In another embodiment, the cell isa cardiac cell, a kidney cell, a hepatic cell, an airway epithelialcell, a muscle cell, a neuronal cell, a glial cell, a microglial cell,an endothelial cell, a mononuclear cell, a tumor cell, a mammalian cell,an insect cell, or a Xenopus oocyte.

[0061] The present invention further provides a compound, previouslyunknown, identified by the screening methods herein. According to oneembodiment, the compound is a peptide, a peptidomimetic, a nucleic acid,a polymer, or a small molecule. The small molecule may be an organic oran inorganic molecule. The small molecule may have a molecular weightless than that of a BCNG protein. According to an embodiment of thepresent invention, the compound is bound to a solid support. In oneembodiment of the present invention, the BCNG protein or theBCNG-related protein is ion channel protein or a protein which is asubunit of an ion channel subunit protein. In one embodiment theBCNG-related protein is a component which is needed to create apacemaker current in and among cells. In an embodiment of the presentinvention, the compound is an agonist or antagonist of ion channelactivity.

[0062] According to an embodiment of the present invention, themodulation is increased ion flow rate or decreased ion flow rate.According to another embodiment, the modulation is increased ionpermissivity or decreased ion permissivity.

[0063] The present invention also further provides a method ofmodulating BCNG protein activity or BCNG-related protein activity in asample, comprising contacting the sample with the provided compound.

[0064] Further, the present invention also provides a method of treatinga condition in a subject which comprises administering to the subject anamount of the provided compound, effective to treat the condition. Thecondition comprises an abnormal condition. The abnormal condition may bea loss of memory, a cardiac condition, a hepatic condition, a problemwith cellular secretions, a pancreatic condition, a pacemaker conditionin brain, or a pacemaker condition in non-neuronal cells.

[0065] The present invention additionally provides a pharmaceuticalcomposition which comprises the provided compound and a pharmaceuticallyacceptable carrier. In an embodiment of this invention, the carrier is adiluent, an aerosol, a topical carrier, an aqueous solution, anonaqueous solution or a solid carrier.

[0066] The present invention also additionally provides a method fortreating a condition in a subject which comprises administering to thesubject an amount of the provided pharmaceutical composition, effectiveto treat the condition in the subject.

[0067] In an embodiment of the present invention, the condition is aneurological, renal, pulmonary, hepatic, or cardiovascular condition.According to an embodiment of this invention, the condition is epilepsy,Alzheimer's Disease, Parkinson's Disease, long QT syndrome, sick sinussyndrome, age-related memory loss, cystic fibrosis, sudden deathsyndrome, hyperalgesia, ventricular or atrial arrhythmias, familialsinus node disease or a pacemaker rhythm dysfunction. In a furtherembodiment of this invention, the subject is a human. Certain additionalmethods for treating diseases in a subject are discussed hereinbelow.Long QT disease is a cardiac disease wherein action potentials lastlonger than they normally should. Sick sinus disease is an acquireddisease (e.g. after atrial fibrilation) wherein the sinus node does notfunction normally.

[0068] The present invention additionally also provides an antibodywhich binds specifically to a BCNG protein or a BCNG-related protein.The present invention further provides a cell capable of producing theantibody. The present invention also provides a method of identifying aBCNG protein or a BCNG related protein in a sample comprising: a)contacting the sample with an antibody under conditions permissive tothe formation of a complex between the antibody and the protein; b)determining the amount of complex formed; and c) comparing the amount ofcomplex formed in step (b) with the amount of complex formed in theabsence of the antibody, the presence of an increased amount of complexformed in the presence of the antibody indicating identification of theprotein in the sample.

[0069] As used herein, the term “BCNG protein” encompasses a proteinhaving an amino acid sequence substantially similar to or identical tomBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4-, hBCNG-1 or hBCNG-2. An example of aBCNG protein is mBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4, hBCNG-1 or hBCNG-2.A BCNG protein may be a homolog of mBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4,hBCNG-1 or hBCNG-2 in a species other than mouse or human. Alternativelya BCNG protein may be another member of the family of BCNG proteins inmouse, human or other mammalian or non-mammalian species. A BCNG proteinmay function as an integral component or subunit of an ion channel. ABCNG protein may be an accessory protein or a non-functional proteinassociated with an ion channel.

[0070] The term “BCNG-related protein” encompasses a protein havingsubstantial homology to at least one functional domain of a BCNG proteinas described herein. (See, Example 3, FIG. 11 and FIG. 13). For example,the hydrophobic core is one such domain (See, Example 3, subsection “TheHydrophobic Core”). Another example of a functional domain is the S4voltage-sensing domain (See, Example 3, subsection “The S4voltage-sensing domain). Still another example of a functional domain isthe cyclic nucleotide binding site (See, Example 3, subsection “Thecyclic nucleotide binding site). Yet another example is the pore domaine(See, Example 3, subsection “The pore”). A BCNG-related protein may thusfunction as an integral component or subunit of an ion channel. ABCNG-related protein may be an accessory protein or a non-functionalprotein associated with an ion channel. A BCNG-related protein isdefined by a sequence or structural homology with a BCNG protein orportion thereof. Thus, a BCNG protein is a BCNG-related protein, but aBCNG-related protein is not limited to BCNG proteins.

[0071] The amino acid sequence of mBCNG-1 is presented as Seq.ID.No.: 2.This sequence has been deposited in the GenBank database and accordedthe GenBank Access-ion Number:AF028737.

[0072] A BCNG protein exhibits substantial sequence similarity tomBCNG-1. A BCNG-related protein exhibits substantial homology orfunctional relatedness to mBCNG-1. Substantial sequence homologyincludes consideration to conserved amino acid substitutions asunderstood by one of skill in the art. Functional relatedness may begleaned from domains or regions of sequence having similarity, separatedby regions with no apparent homology.

[0073] The present invention provides a composition comprising a BCNGprotein or portion thereof, a BCNG-related protein or portion thereof, anucleic acid encoding a BCNG protein or portion thereof, a nucleic acidencoding a BCNG related protein or portion thereof, an antibody to aBCNG protein or portion thereof, an antibody to a BCNG related proteinor portion thereof, an nucleic acid with a sequence antisense to aportion of either a BCNG protein or a BCNG related protein or any otherdescribed compounds of the present invention. The composition mayfurther comprise a carrier. The carrier may be a pharmaceuticallyacceptable carrier.

[0074] As used herein, a “portion thereof” is a sequence. (e.g. am aminoacid sequence or a nucleotide sequence) which comprises less than theentire sequence. For example, in reference to Seq.ID.No.: 2, amino acids35-45 represent a portion thereof.

[0075] As used herein, the term “specifically hybridize” means that anucleic acid probe hybridizes to a nucleic acid sequence havingsubstantial homology with that of the probe. The sequence need not beidentical or unique. However, the sequence must indicate a structural orfunctional relationship between the sequences as is understood in theart.

[0076] Hybridization can distinguish between closely-related anddistantly-related members of a gene family. Reaction conditions can beadjusted to optimize hybridization of one species and minimizehybridization of others.

[0077] For a poorly-matched hybrid, the hybridization is lower and thehybridization curve is displaced towards lower temperatures. When theratio of rate constants (discrimination ratio) for cross-hybridizationand for self-hybridization is plotted against temperature of reaction, asigmoidal curve is obtained. At low temperatures, the ratio is highwhile at higher temperatures (approaching T_(m) −20° C. forperfectly-matched hybrids), the ratio approaches zero.

[0078] The relationship is useful in that it predicts that it should beeasier to distinguish between distantly related sequences by incubatingat low temperatures while it should be easier to distinguish closelyrelated sequences by hybridizing at high temperatures.

[0079] In order, to distinguish between the distantly-related members ofa family of sequences, hybridization should take place at a morepermissive (relaxed) criterion. To detect closely-related members, thehybridization should be at a stringent criterion. A single compromisecriterion will not be effective because, different members of the familyprobably have different discrimination versus temperature curves.Hybridization at a relaxed criterion followed by washing underprogressively more stringent conditions may be useful for detectingdistantly-related members of a family, but is not suitable foridentifying closely-related members. This is probably becausehybridization and washing depend on different parameters. Hybridizationdepends on the nucleation frequency while washing depends on the thermalstability (T_(m)) of the hybrids. Thus, a stringent hybridizationfollowed by a stringent wash is better for detecting closely-relatedmembers of a family than permissive hybridization and a stringent wash.

[0080] The degree of hybridization depends on the degree ofcomplementarity, the length of the nucleic acid molecules beinghybridized, and the stringency of the conditions in a reaction mixture.Stringency conditions are affected by a variety of factors including,but not limited to temperature, salt concentration, concentration of thenucleic acids, length of the nucleic acids, sequence of the nucleicacids and viscosity of the reaction mixture. More stringent conditionsrequire greater complementarity between the nucleic acids in order toachieve effective hybridization.

[0081] A preferred method of hybridization is blot hybridization. SeeSambrook et al. 1989 Molecular Cloning: A Laboratory Manual 2nd Ed. foradditional details regarding blot hybridization.

[0082] As used herein, a nucleic acid probe is a nucleic acid whichspecifically hybridizes to a particular nucleic acid sequence. The probemay be bound nonspecifically to a solid matrix. The nucleic acid probemay be DNA or RNA and can be labeled with a detectable marker. Suchlabeling techniques methods include, but are not limited to,radio-labeling, digoxygenin-labeling, and biotin-labeling. A well-knownmethod of labeling DNA is ³²p using DNA polymerase, Klenow enzyme orpolynucleotide kinase. In addition, there are known non-radioactivetechniques for signal amplification including methods for attachingchemical moieties to pyrimidine and purine rings (Dale, R.N. K. et al.,1973 Proc. Natl. Acad. Sci. USA 70:2238-42), methods which allowdetection by chemiluminescence (Barton, S. K. et al., 1992 J. Am. Chem.Soc. 114:8736-40) and methods utilizing biotinylated nucleic acid probes(Johnson, T. K. et al., 1983 Anal. Biochem. 133:125-131; Erickson, P. F.et al., 1982 J. Immunol. Methods 51:241-49; Matthaei, F. S. et al., 1986Anal. Biochem. 157-123-28) and methods which allow detection byfluorescence using commercially available products. Non-radioactivelabeling kits are also commercially available.

[0083] Nucleic acid amplification is described in Mullis, U.S. Pat. No.4,683,202, which is incorporated herein by reference.

[0084] In a polymerase chain reaction (PCR), an amplification reactionuses a template nucleic acid contained in a sample can use one or moreprobe (“primer”) sequences and inducing agents.

[0085] Suitable enzymes to effect amplification, specifically extensioninclude, for example, E. coli DNA polymerase I, thermostable Taq DNApolymerase, Klenow fragment of E. coli DNA polymerase I, T4 DNApolymerase, other available DNA polymerases, reverse transcriptase andother enzymes which will facilitate covalent linkage of the nucleotidesto polynucleotides which are form amplification products.Oligonucleotide probes (primers) can be synthesized by automatedinstruments sold by a variety of manufacturers or can be commerciallyprepared.

[0086] Solid matrices are available to the skilled artisan. A solidmatrix may include polystyrene, polyethylene, polypropylene,polycarbonate, or any solid plastic material in the shape of test tubes,beads, microparticles, dip-sticks, plates or the like. Additionallymatrices include, but are not limited to membranes, 96-well microtiterplates, test tubes and Eppendorf tubes. Solid phases also include glassbeads, glass test tubes and any other appropriate shape made of glass. Afunctionalized solid phase such as plastic or glass which has beenmodified so that the surface carries carboxyl, amino, hydrazide, oraldehyde groups can also be used. In general such matrices comprise anysurface wherein a ligand-binding agent can be attached or a surfacewhich itself provides a ligand attachment site.

[0087] As used herein, the term “modulation” in reference to modulationof protein activity or ion channel activity refers to the up-regulationor down-regulation of the activity. Up-regulation includes, but is notlimited to increased ion flow in the case of an ion channel.Down-regulation includes, but is not limited to decreased ion flow inthe case of an ion channel. For example, one form of modulation ofactivity would be a channel-blocking protein or compound which inhibitsion flow through the channel, decreasing activity. Alternatively,another form of modulation is a channel-opening protein or compoundwhich facilitates flow through the channel, increasing activity.

[0088] In addition, the nature of the ion flow through a channel may bemodulated. For example, proteins or compounds may alter a channelrefractive to potassium flow to become permissive to potassium flow inaddition to or in place of another ion. The term modulation is also usedto describe the increase or decrease of gene expression. In the practiceof any of the methods of the invention or in the preparation of any ofthe pharmaceutical compositions of the present invention a“therapeutically effective amount” is an amount which is capable ofmodulating the activity or function of a BCNG-related protein.Accordingly, the effective amount will vary with the subject beingtreated, as well as the condition to be treated. The methods ofadministration may include, but are not limited to, administrationcutaneously, subcutaneously, intravenously, parenterally, orally,topically, or by aerosol.

[0089] As used herein, the term “suitable pharmaceutically acceptablecarrier” encompasses any of the standard pharmaceutically acceptedcarriers, such as phosphate buffered saline solution, water, emulsionssuch as an oil/water emulsion or a triglyceride emulsion, various typesof wetting agents, tablets, coated tablets and capsules. An example ofan acceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid®.

[0090] Typically such carriers contain excipients such as starch, milk,sugar, certain types of clay, gelatin, stearic acid, talc, vegetablefats or oils, gums, glycols, or other known excipients. Such carriersmay also include flavor and color additives or other ingredients.

[0091] This invention also provides for pharmaceutical compositionstogether with suitable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions are liquids orlyophilized or otherwise dried formulations and include diluents ofvarious buffer content (e.g., Tris-HCl., acetate, phosphate), pH andionic strength, additives such as albumin or gelatin to preventabsorption to surfaces, detergents (e.g., Tween 20, Tween 80, PluronicF68, bile acid salts), solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulkingsubstances or tonicity modifiers (e.g., lactose, mannitol), covalentattachment of polymers such as polyethylene glycol to the compound,complexation with metal ions, or incorporation of the compound into oronto particulate preparations of polymeric compounds such as polylacticacid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multi lamellar vesicles, erythrocyteghosts, or spheroplasts. Such compositions will influence the physicalstate, solubility, stability, rate of in vivo release, and rate of invivo clearance of the compound or composition.

[0092] Controlled or sustained release compositions include formulationin lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehendedby the invention are particulate compositions coated with polymers(e.g., poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

[0093] When administered, compounds are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive compounds may by required to sustaintherapeutic efficacy. Compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified compounds(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987).Such modifications may also increase the compound's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound adducts less frequently or in lower doses thanwith the unmodified compound.

[0094] Attachment of polyethylene glycol (PEG) to compounds isparticularly useful because PEG has very low toxicity in mammals(Carpenter et al., 1971). For example, a PEG adduct of adenosinedeaminase was approved in the United States for use in humans for thetreatment of severe combined immunodeficiency syndrome. A secondadvantage afforded by the conjugation of PEG is that of effectivelyreducing the immunogenicity and antigenicity of heterologous compounds.For example, a PEG adduct of a human protein might be useful for thetreatment of disease in other mammalian species without the risk oftriggering a severe immune response. The carrier includes amicroencapsulation device so as to reduce or prevent an host immuneresponse against the compound or against cells which may produce thecompound. The compound of the present invention may also be deliveredmicroencapsulated in a membrane, such as a liposome.

[0095] Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside-chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

[0096] Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

[0097] The invention provides nucleic acids comprising cDNA encodingBCNG protein as listed below: Plasmid name ATCC Accession No. Date ofDeposit mBCNG-1 Apr. 21, 1998 mBCNG-2a May 1, 1998 mBCNG-2b May 1, 1998mBCNG-3a May 1, 1998 mBCNG-3b May 1, 1998 hBCNG-1 May 1, 1998 hBCNG-2May 1, 1998

[0098] The above-identified plasmids, provided by the present invention,were deposited with The American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20108-0971, U.S.A. under theprovisions of the Budapest Treaty for the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure.

[0099] Treatment of Diseases or Conditions in a Subject

[0100] The compounds and compositions of the present invention may beadministered to a subject to treat a disease or condition which isassociated with pacemaker function. The compounds and compositionscomprises not only BCNG proteins or BCNG related proteins and nucleicacids encoding the same or portions thereof, but also compoundsidentified by the screening methods of the present invention. Forexample, a compound useful in the present invention may be a peptide, asmall molecule (organic or inorganic), a peptidomimetic, or othercompound as described hereinabove.

[0101] For example, the compounds and compositions of the presentinvention may be useful for treating memory deficits or disorders. Suchmemory disorders or deficits may involve an abnormal pacemaking functionin the cells of the brain and the central nervous system. The memorydisorder or deficit may be due to Alzheimer's disease, Parkinsons'sDisease, or age-related memory loss.

[0102] The present invention provides for a method for treating asensory disorder in a subject comprising administering to the subjectcompounds or compositions of the present invention. Such sensorydisorders include sensory disorders of the eyes (blindness, loss ofvision), of the nose (loss of smell), touch (numbness), and taste (lackof ability to taste).

[0103] In another example, the present invention provides for a methodfor treating a subject suffering from an auditory disorder whichcomprises administering an amount of the compounds or compositions ofthe present invention. It is shown hereinbelow that BCNG isoforms areexpressed in the tissues of the auditory system in signifcant amounts.It may be possible to change the response characteristics of the cellsof the auditory systems by regulation of the BCNG genes in theseauditory tissues and cells. Thus, administration of nucleic acids orproteins (for example via a localized virus vector, or via a liposomecarrying protein) to the subject would treat such an auditory disorder.The auditory disorder may be deafness or loss of hearing.

[0104] In another embodiment, the present invention provides for amethod for treating a subject suffering from a respiratory disordereither due to defects in CNS (central nervous system) areas that controlrespiration or due to defects in the lung, which comprises administeringto the subject an amount of a compound or composition of the presentinvention. Preferably, the compound comprises the BCNG isoform or acompound (e.g. a small molecule) which interacts with the BCNG isoform,which is normally expressed in lung tissue and/or brain nuclei importantfor respiratory control. The respiratory disorder may be Sudden InfantDeath Syndrome, or any difficulty in regular breathing (e.g. shortnessof breath). The repiratory disorder may be asthma.

[0105] The present invention provides a method for the treatment ofdyslexia in a subject which comprises administering to the subject acompound or composition of the present invention in an amount effectiveto treat the dylexia in the subject. The present invention also providesmethods for treatment of attention deficit disorder or learningdisabilities. Learning related disorders may result from abnormalfunctioning (either increased or decreased) of ion channels present inthe thalamus. This region of the brain is considered to be involved inwakefulness, attention and arousal. Disorders involving abnormal statesof such functions may be treatable using the compounds andpharmacuetical compositions of the present invention.

[0106] The present invention provides a method for treating symptoms ofdrug addiction in a subject which comprises administering to the subjecta composition of the present invention to thereby modulate ion channelfunction in the subject and treat the symptoms of drug addiction in thesubject.

[0107] The present invention provides a method for regulating thesecretions of a cell which normally produces secretions in a subjectsuffering from abnormal secretions or lack of secretions which comprisesadministering to the subject a therapeutically effective amount of acompound or composition of the present invention in order to regulatethe secretions of the cell. The compound or composition may regulate theresting phase or the secretion phase of the cell so as to regulate whenthe cell produces secretions.

[0108] The cell may be a pancreatic cell, a liver cell or a spleen cell.The present invention provides a method for regulating reboundexcitation in non-pacemaking cells.

[0109] The BCNG proteins are useful targets for screens for drugs thatare effective in the control of pain and hyperalgesia. Pacemaker typechannels with properties similar to those of the expressed BCNG-1protein have been identified in primary afferent sensory neurons, wherethe channels are activated by prostaglandin E2, a hyperalgesia-inducingagent released during inflammation (Ingram and Williams, 1996). Thechannels have been proposed to play a role in pain perception andhypersensitivity to painful stimuli. The present invention provides amethod for treating pain in a subject which comprises administering tothe subject an amount of the composition of the present invention TheBCNG channel isoforms expressed in cardiac ventricular muscle, includingBCNG-2, are useful targets for screens for drugs that are effective intreating ventricular and/or atrial arrhythmias due to abnormal pacemakeractivity in these tissues. Pacemaker channels with abnormal activationproperties are detected in non-pacemaking regions of the heart,including ventricle, during heart failure (Cerbai et al., 1994, 1997).

[0110] The BCNG gene isoforms expressed in sinoatrial node can provide auseful genetic screen for inherited diseases of cardiac pacemakerfunction such as familial sinus node disease. Certain familial,inherited cardiac diseases are thought to involve defects in pacemakerchannel function in the sinoatrial node (Spellberg, 1971).

[0111] The BCNG isoforms expressed in heart will be useful to screen forimproved drugs that can limit heart muscle damage during episodes ofischemia. Pacemaker channel blockade with the compound ZD 7288 has beenshown to reduce infarct size during myocardial ischemia (Schlack et al.,1998).

[0112] The BCNG-1 channel isoforms will be useful as a screen for drugsthat alter pancreatic function, including compounds that stimulate orinhibit insulin release. It was demonstrated that the human BCNG-1protein is expressed in pancreas (Santoro et al., 1998).

[0113] The BCNG channel isoforms will be useful to screen drugs thatalter function of kidney and liver. BCNG isoforms are expressed in thesetissues where they could contribute to hormone release and ion transportfunctions. Cyclic nucleotide (CGMP)-stimulated activity of a 1 pSchannel, similar to the conductance of the pacemaker channel, has beenreported for renal epithelial cells (Marunada et al, 1991). Liver cellshave been shown to exhibit cation permeable channels coupled to cellularmetabolism (Lidofsky et al. 1997).

[0114] This invention is illustrated in the Experimental Details sectionwhich follows. These sections are set forth to aid in an understandingof the invention but are not intended to, and should not be construedto, limit in any way the invention as set forth in the claims whichfollow thereafter.

[0115] Experimental Details

EXAMPLE 1

[0116] Interactive cloning with the SH3 domain of N-src identifies a newbrain-specific ion channel protein, with homology to cyclicnucleotide-gated channels

[0117] By screening for molecules that interact with the neuronal formof Src tyrosine kinase a novel cDNA was isolated that appears torepresent a new class of ion channels. The encoded polypeptide, mBCNG-1,is distantly related to proteins in the family of the cyclicnucleotide-gated channels and the voltage-gated channels, Eag and H-erg.mBCNG-1 is expressed exclusively in the brain as a glycosylated proteinof approximately 132 kD. Immunohistochemical analysis indicates thatmBCNG-1 is preferentially expressed in specific subsets of neurons inthe neocortex, hippocampus and cerebellum, in particular pyramidalneurons and basket cells. Within individual neurons, the mBCNG-1 proteinis localized to either the dendrites or the axon terminals depending onthe cell type.

[0118] Southern blot analysis shows that several other BCNG-relatedsequences are present in the mouse genome, indicating the emergence ofan entirely new subfamily of ion channel coding genes. These findingssuggest the existence of a novel class of ion channel, which ispotentially able to modulate membrane excitability in the brain andwhich may respond to regulation by cyclic nucleotides.

[0119] Defining signal transduction pathways that contribute to thecontrol of synaptic strength in the brain is an important andlong-sought goal. In an effort to identify the biochemical targets ofSrc-family tyrosine kinases in the central nervous system, the yeasttwo-hybrid system was used to clone proteins that could interact withthe SH3 domain of the neural specific form of Src kinase (Brugge, etal., 1985; Martinez, et al., 1987). As a result of this screening a newprotein, mBCNG-1 (mouse Brain Cyclic Nucleotide Gated-1) was identifiedand isolated.

[0120] mBCNG-1 has been identified and characterized as an ion channelprotein and exhibits sequence homology to voltage-gated potassiumchannels, CNG channels, and plant inward rectifers. Southern blotanalysis suggests that this is the first member of a new family ofproteins. mBCNG-1 is expressed exclusively in the brain and ispreferentially localized to the processes of subsets of neurons in theneocortex, cerebellar cortex and hippocampus. The specific localizationpattern of mBCNG-1 and the potential for a direct interaction withcyclic nucleotides suggest that it may represent a new brain-specificion channel protein that is an important component in the expression ofintercellular and intracellular signaling.

[0121] Results

[0122] Isolation of mBCNG-1. mBCNG-1, a novel cDNA with homology toCNG-related and Eag-related ion channels was initially isolated andidentified by interactive cloning with the N-src SH3 domain in a yeasttwo-hybrid screen. The src gene expresses an alternatively spliced form(N-src or pp60^(src-c) (⁺), which is specific for neuronal cells and hasan increased kinase activity (Brugge, et al., 1985). The N-src proteindiffers from the non-neuronal form (c-src or pp60^(src-c)) by aninsertion of six amino acids in the region corresponding to the Srchomology 3 (SH3) domain of the protein (Martinez et al. 1987). SH3domains are considered modules for protein-protein interaction (Pawson,et al., 1995). Therefore the yeast two-hybrid screen (Fields, et al.,1989; Zervos, et al., 1993) was used to identify brain specific proteinsthat would selectively interact with the N-src SH3 domain.

[0123] The screening of 5×10⁵ independent clones with the N-src SH3 baitresulted in the isolation of a single positively reacting fusion product(pJG-d5). This clone encoded a protein that showed a strong interactionwith the N-src SH3 domain, but no significant interaction with thec-src, fyn, or abl SH3 domains, indicating a specific recognition of theN-src SH3 domain in the yeast two-hybrid system. The sequence analysisof pJG-d5 indicated that it encodes the C-terminal portion of a largerprotein. Overlapping cDNA clones were therefore isolated from a Xgt10library and an open reading frame (ORF) was identified that encodes a910 amino acid polypep tide with a predicted molecular weight of 104 kDa(FIG. 1A). The pJG-d5 insert corresponds to its C-terminal amino acids404-910.

[0124] The N-terminal part of the predicted protein contains anhydrophobic core comprising seven hydrophobic domains (FIG. 1B). Thesedomains show significant homology to the six transmembrane domains(S1-S6) and the pore region (P) of voltage activated K⁺ channels (FIG.1C). In addition to the hydrophobic core, there is a putative cyclicnucleotide binding site (CNBs) in the C-terminal half of the protein(amino acids 472-602, FIG. 1A and FIG. 1D). This cyclic nucleotidebinding site is most closely related to the corresponding region incyclic-nucleotide gated channels (30% similarity). The amino acids thatlie close to the bound cyclic nucleotide in the bacterial catabolitegene activator protein (CAP) are conserved in the N-src interactingprotein, suggesting that the CNBs is functional (Weber et al., 1989). Onthe basis of these features, the newly identified protein was designatedmBCNG-1 (mouse Brain Cyclic Nucleotide Gated-1).

[0125] Among all the known K⁺ channel superfamily genes, the core regionof mBCNG-1 displays the highest amino acid similarity (22%) to thecorresponding region in the mouse Eag protein, whereas the sequencesimilarity to cyclic nucleotide-gated channels is only. 17% in thisregion (distances were determined by the MegAlign program of DNASTAR).The S4 domain of mBCNG-1 has a total of eight positively chargedresidues (two groups of four, separated by a serine), which again makesit more similar to voltage activated K⁺ channels (Sh and eag families)than to cyclic nucleotide-gated channels.

[0126] The putative pore forming region of the mBCNG-1 protein (FIG. 1C)is also most closely related to the corresponding region in Shaker andEag-related channels (30% sequence similarity in either case). However,it contains significant substitutions in two positions that areotherwise highly conserved in voltage activated K⁺ channels: theaspartate residue which follows the GYG triplet is replaced with alanine(position 352) and the serine/threonine residue at −8 from that positionis replaced with histidine (position 344). Similar substitutions arefound in the β-subunit of the retinal CNG-channel, where the positioncorresponding to the aspartate is occupied by a leucine and a lysine isfound at −8 from that position (Chen, et al., 1993). This suggested thatthe mBCNG-1 protein might be incapable of conducting current per se, butmay act in combination with a second not yet identified polypeptide toform a functional heteromultimeric ion channel.

[0127] mBCNG-1 is a 132 kDa Glycoprotein. To characterize the proteinencoded by the mBCNG-1 cDNA (ATCC Accession No. ______) (Seq.ID.No.: 1),antibodies were generated against two separate domains in the predictedcytoplasmic tail: amino acids 594-720 (fusion protein GST-q1; antiserumαq1) and amino acids 777-910 (fusion protein GST-q2; antiserum αq2).Both antisera specifically immunoprecipitated the in vitro translationproduct of the cloned mBCNG-1 sequence.

[0128] In Western blots of mouse brain extracts, both the αq1 and αq2antisera recognized a diffuse band with an apparent molecular mass of132 kDa (FIG. 2A). Complete abolition of the labeling by preadsorbingthe antisera with a GST-fusion protein incorporating both antigenicdomains (GST-d5, amino acids 404-910) indicates it represents the nativemBCNG-1 subunit. Treatment of the brain extract with N-glycosidase Fprior to the Western blotting results in a substantial reduction of themolecular weight of the observed band, which now co-migrates with the invitro translated BCNG-1 product (FIG. 2B).

[0129] Sequence analysis indicates that three N-glycosylation consensussites are present in the mBCNG-1 protein. Among these, Asn 327 ispredicted to lie between transmembrane domain S5 and the pore (P) on theextracellular side of the plasma membrane (FIG. 1A and FIG. 1B). Thissite corresponds to Asn 327 of the cGMP-gated channel from bovine rodphotoreceptors, where it has been demonstrated to be the sole site ofglycosylation (Wohlfart et al., 1992). Together, these data suggestedthat the cloned cDNA sequence encodes the full length product of themBCNG-1 gene and that mBCNG-1 is a N-linked glycoprotein.

[0130] mBCNG-1 is expressed in neurons. Northern blot analysis revealedthe presence of multiple mBCNG-1 transcripts in poly(A)⁺ RNA from thebrain, the most abundant species being 3.4, 4.4, 5.8 and 8.2 kb long(FIG. 3). The 3.4 kb transcript corresponds in size to the cloned cDNA.No expression was detected in the heart, spleen, lung, liver, skeletalmuscle, kidney or testis. The specific expression of the mBCNG-1 proteinwas confirmed by Western blot analysis.

[0131] The cellular localization of mBCNG-1 within the brain wasexamined by in situ hybridization (FIG. 4) and by immunohistochemicalstaining (FIGS. 5A-5F). In both cases, the highest levels of mBCNG-1expression were detected in the cerebral cortex, in the hippocampus, andin the cerebellum.

[0132] In the cerebral cortex, in situ hybridization shows a strongexpression of the mBCNG-1 mRNA layer V pyramidal neuron cell bodies thatare distributed in a continuous line along the neocortex (FIG. 4).Immunohistochemical analysis reveals a strict subcellular localizationof the mBCNG-1 protein within these cells. Staining of the apicaldendrites (FIG. 5A) extends into the terminal branches of these fibersand is particularly intense in layer I, which contains the terminaldendritic plexus of the pyramidal neurons (FIG. 5B).

[0133] A similar expression pattern can be recognized in thehippocampus. Here, the in situ hybridization shows a strong mBCNG-1 mRNAexpression in the pyramidal cell body layer of areas CA1 and CA3 (FIG.4). The labeling in area CA3 is somewhat less prominent than thelabeling in area CA1. At the protein level, the most intense mBCNG-1immunostaining is observed along the hippocampal fissure, in the layercorresponding to the stratum lacunosum-moleculare (FIG. 5C). This layercontains the terminal branches of the apical dendrites of the pyramidalneurons in area CA1 (Raisman, 1965). Further mBCNG-1 immunoreactivity isdetected within the stratum pyramidale of areas CA1 and CA3; thestaining, however, is absent from the pyramidal cell bodies but israther present in the fibers surrounding them (FIG. 5D). These fibersmost likely represent the basket cell plexus associated to pyramidalneurons.

[0134] The immunostaining in the cerebellum also shows a patterncharacteristic of basket cell expression. In the cerebellar cortex,basket cell nerve endings branch and contact the initial segment of thePurkinje cell axon in a distinct structure known as “pinceau” (Palay, etal., 1974). As shown in FIGS. 5E and 5F, these structures are intenselylabeled by the αq1 and αq2. antisera, while the staining excludes thePurkinje cell bodies. Thus, in basket cells” the mBCNG-1 protein appearsto be selectively localized to axons and is particularly enriched in thenerve terminals. An intense labeling of some brainstem nuclei isobserved by in situ hybridization (FIG. 4) and areas of immunoreactivitywere detected in other brain regions, including the olfactory bulb.

[0135] mBCNG-1 defines a new subfamily of K⁺ channel genes. Most of theion channel sequences characterized so far are members of evolutionarilyrelated multigene families. To investigate whether more sequencesrelated to mBCNG-1 exist, mouse genomic DNA Southern blots were analyzedunder various stringency conditions (FIG. 6).

[0136] The probe (B1-T) was designed in the hydrophobic core region ofmBCNG-1, including transmembrane domains S5, P and S6; the repeat regionin the C-terminal portion of the protein was excluded. Reducing thestringency of the hybridization conditions from 8° C. below the meltingtemperature of the B1-T probe (FIG. 6A) to 33° C. below the meltingtemperature (FIG. 6B) resulted in the detection of a number ofadditional hybridization signals in every lane of the blot. None of theknown sequences in the K⁺ channel superfamily has sufficient homology tomBCNG-1 to hybridize under these conditions. This result suggested thatmBCNG-1 is the first known member of a larger group of related genes,which represent a new branch in the voltage-gated K⁺ channelsuperfamily.

[0137] Discussion

[0138] Voltage-gated potassium (VGK) channels constitute a large andstill expanding superfamily of related genes (Strong, et al., 1993;Warmke and Gonetzky, 1994). The most widely used strategy for cloningnew genes in the VGK family has been by homology to a small number ofinitial members (Sh, eag, and slo from Drosophila (Papazian, et al.,1987; Kamb, et al., 1987; Warmke, et al., 1991; Atkinson, et al., 1991);cGMP-channel from bovine retina (Kaupp, et al., 1989). Unfortunately,this approach is not well suited for identifying more divergentsequences. Expression cloning in Xenopus ocytes can circumvent thisproblem, however, this implies a pre-existing or readily detectablephysiological characterization of the channel.

[0139] An alternative cloning strategy that requires no a prioriknowledge of the structure or activity of the target protein is toscreen for K⁺ channels by means of protein-protein interactions. Usingthe SH3 domain of N-src as a bait, a protein, mBCNG-1, was obtained thatappears to constitute a new branch of the K⁺ channel superfamily.mBCNG-1 displays the motifs of a voltage-gated K⁺ channel (sixtransmembrane spanning domains, a highly basic S4, and a P region)(Strong, et al., 1993, Warmke, et al., 1994 and FIGS. 1A-1D). mBCNG-1,despite its similarity to voltage activated K⁺ channel superfamilymembers, with defined by the presence of six transmembrane domains and apore-like region (Warmke, et al., 1994), shows considerable divergencefrom all of the other known sequences. Although the cyclic nucleotidebinding site of mBCNG-1 is most similar to the site present in CNGchannels (30%), the S4 and previous are most closely related to thecorresponding regions in Shaker and Eap. Overall, the highest similarityin the hydrophobic core region is to mouse Eag Protein (22%). Thus,mBCNG-1 appears to constitute a new branch of the K⁺ channelsuperfamily.

[0140] The fusion between an ancestral K⁺ channel and an ancestralcyclic nucleotide binding site is likely to have occurred prior to theevolutionary separation between plants and animals (Warmke, et al.,1994). Divergence from this common ancestor would have led on one handto Eag-related channels and plant inward rectifiers (which maintainedmore of the features of voltage activated K⁺ channels, while showing aprogressive deviation from the original CNBs sequence) and on the otherhand to CNG-channels (which show a higher evolutionary constraint on thecyclic nucleotide binding site, while they have lost voltage activationand K⁺ selectivity). The features of mBCNG-1 suggest that it may haveremained closer to the ancestral molecule that represents theevolutionary link between voltage-gated K⁺ channels and cyclicnucleotide-gated channels.

[0141] The emerging pattern for olfactory and retinal CNG-channels andthe non-consensus sequence of the putative pore forming region ofmBCNG-1 suggests that the lack of detectable electric current followingmBCNG-1 expression in xenopus oocytes is due to mBCNG-1 representing a βsubunit of a heteromultimeric channel (Chen, et al., 1993; Liman, etal., 1994; Bradley, et al., 1994). Indeed the data show the existence ofa number of BCNG-related sequences in the mouse genome, and one or moreof these genes could encode additional subunits required for theformation of an active channel.

[0142] mBCNG-1 protein is expressed only in the brain and in particularin two of the principal classes of neurons within the cerebral,hippocampal and cerebellar cortexes: pyramidal neurons and basket cells.This distribution would be consistent with an in vivo interaction ofmBCNG-1 with N-src, which is also expressed in cerebral and hippocampalpyramidal neurons (Sugrue, et al., 1990). The observed interactionbetween mBCNG-1 and the N-src SH3 domain is intriguing as is itsphysiological relevance and the role of the proline-rich region. Thepossibility that other factors may target the proline-rich region ofmBCNG-1 has also to be considered, particularly in view of the recentlydiscovered WW domains (Sudol, et al., 1996; Staub, et al., 1996).

[0143] The varied subcellular localization of mBCNG-1 (dendritic inpyramidal cells and axonal in basket cells) suggests that mBCNG-1 couldplay different roles in different populations of neurons, perhaps byregulating presynaptic or postsynaptic membrane excitability dependingon the cell type. A similar distribution has been demonstrated for theK⁺ channel subunit Kv 1.2 (Sheng, et al., 1994; Wang, et al., 1994). Kv1.2 forms heteromultimeric K⁺ channels with several other Shaker typesubunits, which have an overlapping yet differential pattern ofexpression, giving rise to a range of conductances with diversifiedfunctional characteristics.

[0144] The presence of mBCNG-1 in the dendrites of hippocampal pyramidalcells is particularly intriguing; cAMP has been shown to be importantfor the establishment of some forms of long-term synaptic potentiationin these cells (Frey, et al., 1993, Bolshakov, et al., 1997; Thomas, etal., 1996). The structural features of mBCNG-1 predict a K⁺ conductingactivity, directly modulated by cyclic nucleotide binding.Interestingly, a current with similar characteristics has been describedin the hippocampal pyramidal neurons of area CA₁ (Pedarzani, et al.,1995), where mBCNG-1 is highly expressed. This current (IQ) is believedto contribute to the noradrenergic modulation of hippocampal activity,by regulating neuronal excitability in response to cAMP levels. mBCNG-1could participate in the formation of the channels responsible for thistype of current.

[0145] Experimental Procedures

[0146] Yeast two hybrid interaction cloning of mBCNG-1. The two-hybridscreen was performed following published procedures (Zervos, et al.,1993); the reagents used included plasmids pEG202, pJG4-5, pJK103 andSaccharomyces cerevisiae strain EGY48 (MATa trpl ura3 his3LEU2::pLexAop6-LEU2).

[0147] The bait was created by subcloning the SH3 domain of N-src inplasmid pEG202, and contains amino acids 83-147 from the mouse N-srcsequence (Martinez, et al., 1987). The cDNA fusion library wasconstructed in plasmid pJG4-5, using poly(A)⁺ RNA from the whole brainof an adult C57BL/6 male mouse; the cDNA was synthesized using randomhexamers and the GIBCO-BRL SuperScript II synthesis kit, according tothe manufacturer's instructions. Only the library constructed from thetwo fractions with an average cDNA size of >1.5 kb (total of 1×10⁶independent clones) was used in the two hybrid screen. Libraryamplification was done in 0.3% SeaPrep agarose (FMC) to avoid changes incomplexity.

[0148] For library screening, Saccharomyces cerevisiae strain EGY48 wasfirst cotransformed with the bait plasmid pEG202-Nsrc and the reporterplasmid pJK103. The resulting strain was maintained under selection forthe HIS3 and URA3 markers, and subsequently transformed with the mousebrain cDNA library in plasmid pJG4-5. This description though is moreaccurate and should be substituted for the transformation mix was grownfor two days in a Ura⁻ His⁻ Trp⁻ glucose medium containing 0.3% SeaPrepagarose (EMC); the cells were then harvested and plated on Ura⁻ His⁻Trp⁻ Leu⁻-galactose. Leu⁺ colonies were screened for β-galactosidaseactivity using a filter lift assay (Breede and Nasmith, 1985).Positively reacting fusion products were isolated and tested forspecificity following retransformation into an independent yeast strain.Fusion product pJGd5 corresponds to the C-terminal part of mBCNG-1(amino acids 404-910; see FIG. 8).

[0149] Full length cloning of mBCNG-1. For the isolation of the 5′ endregion of the mBCNG-1 cDNA, two rounds of PCR were performed on thepJG4-5 library, using nested oligonucleotides derived from the pJG-d5sequence. The downstream primer in the first round was:5′-AGAGGCATAGTAGCCACCAGTTTCC-3′ (Seq. ID. No.: 13) (d5.RL, correspondingto amino acids 456-463 of the mBCNG-1 sequence; see FIG. 8). Thedownstream primer in the second round was:5′-CCGCTCGAGGCCTTGGTATCGGTGCTCATAG-3′ (Seq. ID. No.: 14) (d5.N2),corresponding to amino acids 424-430 of mBCNG-1 and an added XhoI site).The upstream primer was either of two oligonucleotides designed in thepJG4-5 vector sequence: 5′-GAAGCGGATGTTAACGATACCAGCC-3′ (Seq. ID. No.:15) (B42), located 5′ to the EcoRI site in the B42 acidic patch, or:5′-GACAAGCCGACAACCTTGATTGGAG-3′ (Seq. ID. No.: 16) (ter), located 3′ tothe EcoRI site in the ADH terminator.

[0150] PCR cycling was performed as follows: 1× (2 minutes, 94° C.); 25×(45 seconds, 94° C.; 30 seconds, 58° C.; 3 minutes, 72° C.); 1× (10minutes, 72° C.).

[0151] The longest amplification product obtained from this series ofreactions was a 700 bp DNA fragment, which contained amino acids 204-430from the mBCNG-1 sequence (See FIG. 8). This fragment was subcloned,repurified and used as a probe to screen a Mouse Brain cDNA library inγgt10 (CLONTECH, cat. no. ML3000a), in high stringency conditions(hybridization overnight at 65° C. in 50% formamide, 5× SSC (1× SSC=0.15M sodium chloride/0.015 sodium citrate, pH 7), 5× Denhardt's (1×Denhardt's =0.02% Ficoll/0.02% polyvinylpirrolidone/0.02% bovine serumalbumin), 0.5% SDS, 100 mg/ml salmon sperm DNA. Washing: 10 minutes,room temperature in 2× SSC/0.1% SDS, followed by twice 30 min at 65° C.in 0.2× SSC/0.1% SDS.

[0152] Positively reacting clones were further screened by PCR, usingoligonucleotide d5.RL (Seq. ID. No.: 13) as a downstream primer. Theupstream primer was either of the two following vector oligonucleotides:5′-GAGCAAGTTCAGCCTGGTTAAGTCC-3 (Seq. ID. No.: 17) (15′.N2), located 5′to the EcoRI site in the γgt10 sequence, or5-GTGGCTTATGAGTATTTCTTCCAGGG-3′ (Seq. ID. No.: 18) (13′.N2), located3‘to the EcoRI site’. PCR cycling was performed as described above.

[0153] The resulting products were subcloned and sequenced. The longestextension contained amino acids 1-463 of the mBCNG-1 sequence (See FIG.8); the overlapping region of this insert with the insert contained inclone pJG-d5 (amino acids 405-463) includes a Bgl II site, which wasused to join the 5′ and 3′ fragments of the mBCNG-1 cDNA in plasmidpSD64TF for expression studies.

[0154] In vitro transcription (MESSAGE MACHINE, Ambion, Austin, Tex.)and translation in vitro Express, Stratagene.

[0155] Northern/Southern Blot Hybridization

[0156] PCR-generated cDNA fragments corresponding to the indicated aminoacids 6-131 (γgt10-derived) 5′ sequence) and 594-720 (pJG-5 derived 31sequence) were used to probe a Multiple Tissue Northern Blot (CLONTECH,7762-1).

[0157] For Southern blots, a Mouse Geno-Blot (CLONTECH, 7650-1) wasprobed using a PCR generated cDNA fragment (B1-T) corresponding to aminoacids 270-463 of the mBCNG-1 sequence, as described (Sambrook, 1989).Blots were hybridized at 65° (5× standard saline citrate 1×SSC=0.15Msodium chloride/0.015 M sodium citrate, pH7 buffer in aqueous solution)and washed as described in figure legends. Washings conditions were asindicated. The melting temperature (TM) for the B1-T probe wascalculated according to the formula Tm=81.5° C.+16.6 (log M)+0.41 (%GC)−(675/L), where M is the cation concentration and L is the probelength in base pairs.

[0158] Antibody production extracts and Immunochemistry and in situhybridization. The Glutathione S-transferase (GST)-fusion proteins werecreated by subcloning the q1 (corresponding to amino acids 594-720 ofthe mBCNG-1 protein) or q2 (corresponding to amino acids 777-910 of themBCNG-1 protein) (see FIG. 1A) in plasmid pGEX-lombole (Pharmacia),followed by induction and purification of essentially as described(Frangioni and Neel, 1993). Fusion proteins were eluted in phosphatebuffered saline (PBS) and injected into rabbits as a 1:1 suspension withFreund adjuvant (Pierce). Antisera were prepared and tested essentiallyas described (Grant, et al., 1995).

[0159] For Western Blot analysis, mouse brain extracts were separated ona 10% SDS-PAGE and electroblotted to PVDF membranes (Immobilon-P,Millipore) as described (Grant, 1995). Blocking and antibody incubationswere done in TBST (10 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20)+2%BSA. The αq1 and αq2 antisera were used at a 1:1000 dilution. Secondaryanti-rabbit antibodies coupled to alkaline phosphatase (Bio-Rad) wereused at a 1:5000 dilution, and the bands were visualized by incubationin NBT\BCIP (Boehringer Mannheim). Total brain extracts were prepared asdescribed (Grant, 1995). For N-glycosidase treatment, 2% SDS was addedto the extract and the proteins denatured by boiling for 10 min;reactions were carried out in 50 mM NaP (pH 7.2), 25 mM EDTA, 0.5%Triton-X100, 0.2% SDS, 1 mg/ml protein and 20 U/ml N-glycosidase F(Boehringer) for 1 hr at 37° C.

[0160] For immunohistochemistry, 20 μm cryostat sections of mouse brain(fixed in 4% paraformaldehyde/PBS), quenched in 50 mM NH₄Cl/PBS, wereblocked (10% goat serum, 0.1% goat serum, 0.1% saponin in PBS) and thenexposed to αg1 or αq2 antisera (diluted 1:400 in blocking solution).After washing in PBS+0.1% saponin, sections were incubated withCy3-conjugated goat anti-rabbit F(ab′) 2 fragments (JacksonImmunoreasearch Labs) diluted 1:200 in blocking solution.

[0161] In situ hybridization was performed essentially as described(Mayford et al., 1995) using oligonucleotide probes labeled by 3¹tailing with using³⁵ (S) thio-dATP and terminal transferase (BoehringerMannheim) to a specific activity of 5×10⁸ cpm/μg. Hybridizations werecarried out at 37° C. Slides were washed at 60° C. in 0.2× SSC andexposed to film for 2 weeks.

EXAMPLE 2 Identification of a Family of BCNG Genes

[0162] Introduction

[0163] The original sequence in the BCNG family (mBCNG-1) was isolatedfrom a mouse brain cDNA library using yeast two-hybrid interactioncloning with the n-Src tyrosine kinase as a bait as described inExample 1. The DNA and amino acid sequences of this protein are Seq. ID.No.:1 and Seq. ID. No.:2 respectively.

[0164] Three additional mouse and two human cDNA clones encoding regionshomologous to mBCNG-1 (ATCC Accession No. ______) were isolated. PartialcDNA clones representing two of the mouse genes ([mBCNG-2 (ATCCAccession Nos. ______ and ______] and mBCNG-3 [ATCC Accession Nos.______ and ______]) were isolated while screening for full lengthmBCNG-1 products, and a fourth mouse gene (mBCNG-4) as well as two humangenes (hBCNG-1 [ATCC Accession No. ______] and hBCNG-2 [ATCC AccessionNo. ______] were identified following an EST database homology search,using the protein sequence of mBCNG-1 (Seq.ID.NO.: 2) as a query.Further extensions of the identified cDNA clones were subsequentlyobtained by library screening or RT-PCR cloning. A schematicrepresentation of the mouse and human BCNG sequences identified ispresented in FIGS. 7A-7B.

[0165] The three additional mouse proteins described hereined below areclosely related to each other, having a sequence similarity of 84-88%,but are very distantly related to all other known members of thepotassium channel superfamily, including Eag-related channels (22%similarity) and cyclic nucleotide-gated channels (17% similarity).

[0166] Northern blot analysis showed individual patterns of tissuedistribution for each of these clones (see FIG. 9). The expression ofmBCNG-1 appears to be restricted to the brain (FIG. 9A), whereas mBCNG-2(FIG. 9B) and mBCNG-3 (FIG. 9C) are expressed in the brain as well as inthe heart. Hybridization signals for mBCNG-3 are also detected in polyA⁺RNA from skeletal muscle and lung.

[0167] The distinct sequences and tissue distributions of these clonesreveals that the BCNG clones represent a family of ion channel proteins,with characteristic voltage sensing and cyclic nucleotide bindingmotifs, that are predominantly located in heart and brain. TABLE I_EST_CLONES_IDENTIFIED_(—) BY_HOMOLOGY_TO_mBCNG-1_(—) GeneBank IMAGEGenome Trivial Probable Accession consortium systems Name Identitynumber cDNA ID ID M41-EST 3′ of AA023393 456380 mBCNG-2 M28-EST 3′ ofAA238712 693959 cd-22017 mBCNG-4 H61-EST 3′ of N72770 289005 hBCNG-2H57-EST 3′ of H45591 176364 hBCNG-1

[0168] Predicted Amino Acid Sequence of the Conserved BCNG ChannelFamily. The deduced, integrated amino acid sequences obtained for themBCNG-2 (Seq.ID.No.:6), mBCNG-3 (Seq.ID.No.:10) and mBCNG-4 (Seq.ID.No.:12) encoded proteins are shown in FIGS. 8A-8B, and were aligned to thefull length sequence of mBCNG-1 (Seq.ID.No.:2) (Santoro et al., 1997)(GenBank Accession No.: AF028737). All of the identified sequences(except mBCNG-4) contain the motifs of a voltage-gated potassium channel(Jan and Jan, 1997). Thus, they appear to encode for channel subunitswith an intracellular amino terminus, six putative transmembranespanning domains (S1-S6), and a long cytoplasmic carboxy terminus. TheS4 domain, which serves as the voltage sensor in other voltage-gatedchannels, contains 9 positively charged basic residues, more so than anyother voltage-gated channel. In addition, the three clones contain ahighly conserved pore-forming P region that links the S5 and S6transmembrane segments. This P loop is homologous to the P regions ofvoltage-gated K channels and in particular contains the K channelsignature sequence triplet, GYG, suggesting that the clones will encodea K selective ion channel (Heginbotham et al., 1994).

[0169] The long cytoplasmic tail of the BCNG proteins is predicted tocontain a stretch of 120 amino acids that is homologous to the cyclicnucleotide binding (CNB) sites of cAMP- and cGMP-dependent proteinkinases and the catabolite activating protein, CAP, a bacterial cAMPbinding protein (Shabb and Corbin, 1992). The BCNG cyclicnucleotide-binding domains are most similar to the binding domains ofthe cyclic nucleotide-gated channels involved in visual and olfactorysignal transduction (Zagotta and Siegelbaum, 1996). Although othermembers of the voltage-gated channel family have been reported tocontain CNB sites, these putative sites lack many of the key conservedresidues found in functional cyclic nucleotide-binding proteins (Tibbset al., 1998). Strikingly, these key residues are conserved in the BCNGchannel family, suggesting that these binding sites are likely tofunction.

[0170] Certain pacemaker channels can be regulated by PKAphosphorylation, such as the cardiac Purkinje fiber channel (Chang etal., 1991), whereas other pacemaker channels appear to be directlyregulated by cAMP, such as the sino-atrial node channel (DiFrancesco andTortora, 1991). It is interesting that mouse (and human) BCNG-1 andBCNG-2 contain a serine residue in their cytoplasmic carboxy terminusthat lies within a consensus site for PKA phosphorylation (FIG. 8B,arrow). mBCNG-4 does not contain this site, providing a potentialexplanation for the different modulatory properties of channels indifferent tissues. What is particularly striking about this potentialphosphorylation site is that it lies within the C-helix of the cyclicnucleotide binding site, a region that forms a key part of the ligandbinding pocket (Weber and Steitz, 1987). Studies on rod and olfactorycyclic nucleotide-gated channels previously showed that the C-helixplays an important role in ligand-selectivity and the efficacy ofligand-gating (Goulding et al., 1994; Varnum et al., 1995). Thus thephosphorylation of this serine residue might influence the efficacy withwhich cyclic nucleotides modulate the gating of certain pacemakerchannels.

[0171] The mouse proteins are closely related to each other, having asimilarity of 84-86% (FIG. 7C). Notably, mBCNG-2 and mBCNG-3 are moreclosely related to each other (89% similar) than either is to mBCNG-1.As far as a limited alignment could show (see legend to FIG. 7), mBCNG-4appears to be the most distantly related protein in the group.

[0172] Cloning of Two Human BCNG Genes. The high degree of similaritybetween H57-EST and mBCNG-1 suggested that this EST likely representedthe 3′ end region of the human homolog of mBCNG-1 (designated hBCNG-1).Based on this assumption, PCR oligonucleotide primers were synthesizedin order to amplify hBCNG-1. (See FIG. 7). One primer consisted of asequence in the 5′ end of the mouse BCNG clones (oligo MB1-3[Seq.ID.No.: 26]) and the second primer was based on a sequence in theH57-EST (oligo H57.C [Seq.ID.No.: 27]). (See FIG. 7). A single, strongRT-PCR product of the predicted length was obtained using human brainpolyA+ RNA. No band was obtained from human heart polyA+ RNA. Uponcompletion of the sequencing of the original EST clone and of the RT-PCRproduct, 2247 bp of the hBCNG-1 sequence was obtained (Seq.ID.No.:3)(see FIG. 7).

[0173] The predicted amino acid sequence of the encoded hBCNG-1 protein(Seq.ID.No.: 4) is shown in FIG. 8. Remarkably, the 308 amino acid-longcore region of the hBCNG-1 protein, extending from the S1 through the S6transmembrane segments, is 100% similar to mBCNG-1.

[0174] The H61-EST sequence showed marked sequence similarity to mBCNG-2and could, in fact, encode the human homolog of the mBCNG-2 protein.Accordingly, a sequence within the H61-EST clone was used to probe ahuman brain γgt10 cDNA library. (See FIG. 7). Combining the sequences ofthe H61-EST clone and of the γgt10 clones, 1792 bp. of the hBCNG-2sequence was obtained (Seq.ID.No.: 7) (See FIG. 7). The predicted aminoacid sequence of the encoded hBCNG-2 protein is shown in Seq.ID.No.:8.(See FIG. 8). The 308 amino acid-long core region of the hBCNG-2 proteinis 98% similar to mBCNG-2 (FIG. 7C).

[0175] Tissue Distribution of BCNG mRNA Expression. Northern blotanalysis showed individual patterns of tissue distribution for each ofthe identified clones and a high correspondence in the transcript andlocalization patterns between homologous mouse and human clones (FIGS. 9and 10). While the expression of mBCNG-1 appears to be restricted to thebrain (FIG. 9, and Santoro et al. 1997), mBCNG-2 and mBCNG-3 areexpressed in the brain as well as in the heart (FIG. 9). Hybridizationsignals for mBCNG-3 are also detected in polyA+ RNA from skeletal muscleand lung. A distinct pattern of tissue distribution is revealed formBCNG-4, which appears to be mainly expressed in the liver, but is alsopresent in brain, lung and kidney (FIG. 9D).

[0176] The homologous mouse and human BCNG genes are likely to befunctionally similar since they exhibit very similar patterns of tissueexpression as revealed by the Northern blot analysis. FIG. 10 shows thata probe designed within the hBCNG-1 sequence recognized four transcriptsin human brain polyA+ RNA. This pattern is very similar to that seen inthe Northern blot of mBCNG-1 (FIG. 9 and Santoro et al. 1997). Weakhybridization signals are also detected for hBCNG-1 in human muscle andpancreas. Northern blot analysis using a probe based on the hBCNG-2sequence showed an expression pattern which is highly consistent withthe expression pattern of mBCNG-2 (FIG. 10; compare with FIG. 9). Anabundant 3.4 kb transcript is detected in the brain and the sametranscript is also present in the heart.

[0177] The analysis of the distribution of mBCNG-1 within the mousebrain (Santoro et al. 1997) revealed that the highest expression ofmBCNG-1 occurs in the cortex, hippocampus and cerebellum. Moreover, themBCNG-1 protein is specifically localized to the apical dendrites ofpyramidal neurons as well as to the axon terminals of basket cellswithin these regions. (See, Example 1). Northern blot analysis of thehBCNG-1 mRNA distribution within different brain regions also showed adifferential expression of the gene, with the highest levels present incortical structures (hippocampus and amygdala; FIG. 10). hBCNG-2 shows amore uniform level of high expression in all brain structures,suggesting a more ubiquitous role. In particular, the stronghybridization signal in corpus callosum-derived RNA may indicateexpression of hBCNG-2 within glial cells.

[0178] Experimental Procedures

[0179] Library screening and RT-PCR cloning. Standard manipulations ofEscherichia coli, lambda phage and nucleic acids, including recombinantDNA procedures, were performed essentially as described (Sambrook et al.1989).

[0180] Cloning of mBCNG-2. From the nested PCR reactions performed onthe pJG4-5 library (see Example 1, Full-length cloning of mBCNG-1) anamplification product was isolated, that had a sequence similar but notidentical to the expected mBCNG-1 sequence. It was thus inferred that itrepresented a different gene, closely related to mBCNG-1, which wasdesignated mBCNG-2. The identified fragment (“dA”) encoded amino acids234-430 from the mBCNG-2 sequence (numbering according to mBCNG-1, seeFIG. 8)

[0181] Next performed was a series of RT-PCR reactions on polyA⁺ RNAderived from mouse brain and heart, using oligos:5′-TGGGAAGAGATATTCCACATGACC-3′ (Seq. ID. No.: 19) (7.SEQ1, correspondingto amino acids 270-277 of the mBCNG-1 sequence; see FIG. 8) as anupstream primer, and oligo d5.RL (Seq. ID. No.: 13) as a downstreamprimer. A 600 bp product was obtained from heart polyA⁺ RNA, subcloned,sequenced and shown to be identical to mBCNG-2. PCR cycling: 1× (2minutes, 94° C.); 25× (50 seconds, 94° C.; 4.0 seconds, 52° C.; 1.5minute, 72° C.); 1× (10 minutes, 72° C.).

[0182] The Clontech Mouse Brain γgt10 library was screened at highstringency (see Example 1, Full-length cloning of mBCNG-1), with a PCRprobe derived from the mBCNG-2 sequence (probe “dA”) using oligos:5′-TACGACCTGGCAAGTGCAGTGATGCGC-3′ (Seq. ID. No.: 20) (ASEQ2,corresponding to amino acids 278-286 of the mBCNG-2 sequence, numberingaccording to mBCNG-1; see FIG. 8) as an upstream primer, and5′-AGTTCACAATCTCCTCACGCAGTGGCCC-3′ (Seq. ID. No.: 21) (HRL.2,corresponding to amino acids 444-452 of the mBCNG-2 sequence, numberingaccording to mBCNG-1; see FIG. 8) as a downstream primer.

[0183] Positively reacting clones were further screened by PCR, usingoligonucleotide: 5′-CTGGTGGATATATCGGATGAGCCG-3′ (Seq. ID. No.: 22)(BE-ASE, corresponding to amino acids 262-269 of the mBCNG-2 sequence,numbering according to mBCNG-1; see FIG. 8) as a downstream primer andeither of the two lambda derived oligonucleotides (15′.N2 (Seq. ID. No.:17) or 13′.N2 (Seq. ID. No.: 18)) as an upstream primer (see Example 1.,Full length cloning of mBCNG-1). The clones yielding the longestextension products were subcloned and sequenced, thus obtaining theN-terminal part of the mBCNG-2 sequence up to amino acids 304 (numberingaccording to mBCNG-1; see FIG. 8).

[0184] After obtaining the sequence for EST-M41, a further round ofRT-PCR reactions was performed both on mouse brain and heart polyA⁺ RNA,using oligonucleotides: 5′-CAGTGGGAAGAGATTTTCCACATGACC-3′ (Seq. ID. No.:23) (B123, corresponding to amino acids 269-277 of the BCNG sequences,numbering according to mBCNG-1; see FIG. 8) as an upstream primer, and5′-GATCATGCTGAACCTTGTGCAGCAAG-3′ (Seq. ID. No.: 24) (41REV,corresponding to amino acids 590-598 of the mBCNG-2 sequence, numberingaccording to mBCNG-1; see FIG. 8) as a downstream primer. Extensionproducts of the expected length were obtained from both RNApreparations, subcloned and sequenced, linking the γgt10 derived 5′fragment and the EST derived 3′ fragment of mBCNG-2.

[0185] PCR cycling was performed as follows: 1× (2 minutes, 94° C.); 25×(45 seconds, 94° C., 30 seconds, 55° C.); 2 minutes, 72° C.); 1× (10min, 72° C.).

[0186] Clonina of mBCNG-3. From the γgt10 library screen for mBCNG-2(see above) one positively reacting clone was obtained (#15) whichappeared to give a consistently weaker hybridization signal. This insertwas amplified with oligonucleotides 15.N2 (Seq. ID. No.: 17) and 13.N2(Seq. ID. No.: 18), subcloned, sequenced and shown to represent a thirdBCNG-related sequence, different both from mBCNG-1 and mBCNG-2, whichwas called mBCNG-3. The identified fragment encoded the N-terminal partof the mBCNG-3 sequence up to amino acid 319 (numbering according tomBCNG-1; see FIG. 8).

[0187] After obtaining the sequence for EST-M28, an RT-PCR was performedboth on mouse brain and heart polyA+ RNA using oligonucleotide B123 asan upstream primer, and degenerate oligonucleotide:5′-CACCKCRTTGAAGTGGTCCACGCT-3′ (Seq. ID.

[0188] No.: 25) (28REV, corresponding to amino acids 554-561 of the BCNGsequences, numbering according to mBCNG-1; see FIG. 8) as a downstreamprimer. Extension products of the expected length were obtained fromboth RNA preparations, subcloned and sequenced. Both representedextension of the mBCNG-3 sequence, as determined by an overlap with theknown 3′ end of the γgt10 #15 clone. PCR cycling was performed at: 1× (2minutes, 94° C.); 25× (45 seconds, 94° C., 30 seconds, 55° C., 2minutes, 72° C.); 1× (10 minutes, 72° C.).

[0189] Cloning of hBCNG-1. After obtaining the sequence for EST-H57, anRT-PCR reaction was performed on human brain polyA⁺ RNA, usingoligonucleotides: 51-ATGTTCGGSAGCCAGAAGGCGGTGGAG-3′ (Seq. ID. No.: 26)(MB1-3, corresponding to amino acids 102-110 of the BCNG sequences,numbering according to mBCNG-1; see FIG. 8) as an upstream primer, and5′-CAGCTCGAACACTGGCAGTACGAC-3′ (Seq. ID. No.: 27) (H57.C, correspondingto amino acids 537-544 of the hBCNG-1 sequence, numbering according tomBCNG-1; see FIG. 8) as a downstream primer. A single extension productof the expected length was obtained, subcloned, sequenced, and shown torepresent the 5′ extension of the hBCNG-1 clone.

[0190] PCR was performed as follows: 1× (2 minutes, 94° C.); 25× (45seconds, 94° C., 20 seconds, 58° C., 3 minutes, 72° C.); 1× (10 minute,72° C.).

[0191] Cloning of hBCNG-2. After obtaining the sequence for EST-H61, aPCR probe was made using oligonucleotides: 5′AACTTCAACTGCCGGAAGCTGGTG3′(Seq. ID. No.: 28) (H61.A, corresponding to amino acids 452-459 of thehBCNG-2 sequence, numbering according to mBCNG-1; see FIG. 8) as anupstream primer, and 5′GAAAAAGCCCACGCGCTGACCCAG3′ (Seq. ID. No.: 29)(H61.F, corresponding to aa 627-634 of the hBCNG-2 sequence, numberingaccording to mBCNG-1; see FIG. 8) as a downstream primer on the EST-H61DNA. This fragment was used to screen a Human Brain Hippocampus cDNAlibrary in γgt10 (CLONTECH, cat. no. HL 3023a), in high stringencyconditions (see above). Positively reacting clones were further screenedby PCR, using oligonucleotide: 5′CACCAGCTTCCGGCAGTTGAAGTTG3′ (Seq. ID.No.: 30) (H61.C, corresponding to amino acids 452-459 of the hBCNG-2sequence, numbering according to mBCNG-1; see FIG. 8) as a downstreamprimer and either of oligonucleotides 15′.N2 (Seq. ID. No.: 17) or13′.N2 (Seq. ID. No.: 18) as an upstream primer. The clones yielding thelongest amplification products were subcloned and sequenced, thusobtaining the N-terminal region of the hBCNG-2 sequence up to aa 587(numbering according to mBCNG-1; see FIG. 8).

[0192] Northern blots. For mouse gene expression studies, a MouseMultiple Tissue Northern Blot (CLONTECH, cat. no. 7762-1) was probedwith the following PCR products: For mBCNG-1, probe “q0”, obtained usingoligos q0.5′ (5′GCGAATTCAAACCCAACTCCGCGTCCAA3′) (Seq. ID. No.: 31) andq0.3′ (5′CCTGAATTCACTGTACGGATGGAT3′) (Seq. ID. No.: 32). Amplificationproduct corresponding to aa 6-131 of the mBCNG-1 sequence (see FIG. 7and FIG. 8). For mBCNG-2, probe “dA”, obtained using oligos ASEQ2/HRL.2(see above). For mBCNG-3, probe “15-7”, obtained using oligos15.N2/13.N2 (see above); amplification performed directly on lambdaphage DNA (clone #15). For mBCNG-4, probe “M28” was obtained as agel-purified EcoRI/BglII restriction fragment (400 bp) from the EST-M28DNA. Fragment corresponding to amino acids 529-607 of the mBCNG-4sequence (numbering according to mBCNG-1; see FIG. 8), plus 180nucleotides of the mBCNG-4 3′UTR (untranslated region; see Seq. ID. No.:11).

[0193] For human gene expression studies, a Human Multiple TissueNorthern Blot (CLONTECH, cat. no. 7760-1) or Human Brain Multiple TissueNorthern Blot (CLONETECH, cat. no. 7750-1) was probed with the followingPCR products: For hBCNG-1, probe H57, obtained using oligos H57.A(5′GTCGTACTGCCAGTGTTCGAGCTG3′)(Seq. ID. No.: 33) and H57.B(5′GGTCAGGTTGGTGTTGTGAAACGC3′) (Seq. ID. No.: 34). Fragmentcorresponding to aa 537-800 of the hBCNG-1 sequence (numbering accordingto mBCNG-1; see FIG. 8). For hBCNG-2, probe “H61”, obtained using oligosH61.A (Seq. ID. No.: 28) and H61.F (Seq. ID. No.: 29) (see above).

[0194] Hybridizations were all performed in ExpressHyb solution for 1hour, 68° C., as indicated in the manufacturer's Protocol Handbook.Washing was performed as follows: 10 minutes, room temperature in2×SSC/0.1% SDS, followed by twice 30 minutes, 65° C. in 0.2×SSC/0.1%SDS. Filters were stripped between subsequent hybridizations by boilingfor 5 min in 0.5% SDS/H₂O.

[0195] Sequence alignments and EST database search. Alignments anddistance calculations were all performed with MegAlign (DNASTAR) on theindicated peptide sequences.

[0196] The EST database search was performed with BLAST (NCBI), usingthe mBCNG-1 polypeptide sequence (amino acids 1-720, to avoid theglutamine repeat present in the C-terminal region of the protein) andthe TBLASTN program.

EXAMPLE 3 Physiological and Pharmacological Significance of Mouse andHuman BCNG Channel Genes.

[0197] Introduction

[0198] The unique structural features and the tissue distribution of thepredicted proteins of the BCNG gene family suggested that they encodethe pacemaker current (variously called Ih, If or Iq) of the heart andbrain. Alternatively, it was suggested that it might be a component ofother—perhaps unidentified—ionic current(s) that are important incardiac renal, hepatic and central nervous system function.

[0199] The unique structural features of the predicted BCNG proteins(the unusual ion conducting pore (P) domain, the highly conserved cyclicnucleotide binding (CNB) site and the highly conserved and highlycharged S4 voltage sensor) indicated that they may be susceptible tomultiple drug intervention strategies that target the pore, the cyclicnucleotide binding site and the voltage-dependent gating apparatus.

[0200] Analysis And Predicted Structure and General Features of the BCNGProteins. The predicted amino acid sequence of the BCNG genes revealedthat they are members of the voltage-gated ion channel superfamily.Specifically, the BCNG proteins show similarities to the superfamily ofchannels that includes the voltage-gated K⁺ channels (Pongs, et al.,1995) and the cyclic nucleotide-gated channels, non-selective cationchannels that are permeable to Na, K⁺ and Ca (Zagotta and Siegelbaum,1996). As shown schematically in FIG. 11, the BCNG proteins arepredicted to have six transmembrane spanning α-helices with cytoplasmicN and C termini, a highly basic fourth transmembrane domain (S4) andpore (p) region. Each of these motifs are found in the members of thevoltage-gated K⁺ family. In addition, the BCNG proteins have a wellconserved cyclic nucleotide binding site in the C-terminus. Although ahomologous motif is found in some of the voltage-gated K⁺ channels, thecyclic nucleotide binding sites in those channels are not well conservedand there is little evidence that the binding sites are functional.Indeed, the cyclic nucleotide binding site of the BCNG channels is mosthomologous to the sites found in cyclic nucleotide gated channels whichuse the binding of cyclic nucleotides to drive their activation.Furthermore, while the P loops of the BCNG channels are homologous tothose found in voltage activated K⁺ channels and cyclic nucleotide gatedchannels, there are several non-conservative changes in the amino acidsequence that are likely to yield ion conduction properties that areunique to the BCNG channels. Thus, the BCNG channels appear distinctfrom all previously identified channels in a number of ways whichsuggests that they have distinct physiological and pharmacologicalproperties. These similarities and dissimilarities in the sequences ofthe voltage-gated K⁺ channels, cyclic nucleotide-gated channels and theBCNG channels and the predicted consequences for BCNG channel functionalproperties are discussed more extensively below.

[0201] Results

[0202] The Hydrophobic Core. The core of BCNG channels is predicted tohave six transmembrane (α-helical sequences (S1-S6) and a pore forming Ploop. This assignment is homologous to a single subunit of thetetrameric K⁺ channels (and tetrameric cyclic nucleotide-gated channels)or a single repeat in the pseudo tetrameric Na and Ca channels. Thishomology suggests that the BCNG channels are members of thevoltage-gated K⁺ channel superfamily (which also includes thevoltage-independent but structurally homologous cyclic nucleotide-gatedchannels). It is likely that BCNG channels are composed of four suchpolypeptides in a hetero or homomultimeric structure as is seen for thevoltage-gated K⁺ channels and the cyclic nucleotide-gated channels (Chenet al., 1993; Bradley et al., 1994; Liman and Back, 1994; Lin et al.,1996). However, mBCNG-1 shows considerable divergence from all otherknown K⁺ channel and cyclic nucleotide-gated channel sequences. As notedabove, the highest homology in the hydrophobic core region is to mouseEag (22% amino acid similarity)—a voltage-gated K⁺ channel that has adegenerate and probably non-functional cyclic nucleotide binding siteWarimke and Gancleky. Over this core region, mBCNG-1 shows 17% identityto the voltage independent cyclic nucleotide-gated channels.

[0203] In contrast, the proteins that are predicted to be encoded by theBCNG genes show high homology to each other (>80%, see Examples 1 and2). Indeed, mouse BCNG-1 and human BCNG-1 are identical over the coreregion. Similarly, mBCNG-2 and hBCNG-2 are 98% identical over the coreregion. Thus, the BCNG family of genes appears to constitute a newbranch of the K⁺ channel superfamily which could be regulated by cyclicnucleotide binding. The presence of a gene family with members showingsuch sequence conservation strongly suggests important biologicalfunction.

[0204] The S4 voltage-sensing domain. The presence of positively chargedarginine and lysine residues at every third position in the fourthtransmembrane helix is a signature sequence of voltage-dependent gating(Hille, 1992; Catterall, 1992, see FIG. 12). In contrast, in thevoltage-independent cyclic nucleotide-gated channels, the S4 isdegenerate with some of the positively charged residues being replacedby negatively charged acidic amino acids or being out of the triadrepeat frame. These changes have reduced the net positive charge in theS4 of the cyclic nucleotide-gated channels to 3-4. This introduction ofnegatively charged residues may underlie the reason that the cyclicnucleotide-gated channels no longer respond to voltage. However, it isalso possible that voltage-sensitivity may have been lost as a result ofsome other structural change in the cyclic nucleotide-gated channels andthe divergence in S4 structure is simply a reflection of the loss ofevolutionary pressure to retain the positive charges.

[0205] The S4 of BCNG channels are most closely related to thecorresponding regions in the voltage-gated K⁺ channels Shaker and eag,albeit poorly (mBCNG-1 is 30% homologous to the S4 of Shaker and eag).Despite an interruption by the inclusion of a serine in place of anarginine in the S4 of BCNG channels, the BCNG sequence contains morepositively charged residues than any other member of the voltage-gatedK⁺ channel superfamily (see FIG. 12). The S4 domain of BCNG-1 has up tonine positively charged residues (one group of five and one group offour separated by a serine in place of one other arginine), which againmakes it more similar to voltage activated K⁺ channels (Sh and eagfamilies) than to cyclic nucleotide-gated channels. The retention ofsuch a highly charged S4 strongly suggests that the gating of thesechannels are voltage-sensitive.

[0206] The cyclic nucleotide binding site. Cyclic nucleotides regulatethe activity of a diverse family of proteins involved in cellularsignaling. These include a transcription factor (the bacterialcatabolite activating protein, CAP), the CAMP- and cGMP-dependentprotein kinases (PKA and PKG) and the cyclic nucleotide-gated (CNG) ionchannels involved in visual and olfactory signal transduction (Shabb andCorbin, 1992; Zagotta and Siegelbaum, 1996). Despite obvious divergenceamong the effector domains of these proteins, the cyclic nucleotidebinding sites appear to share a common architecture. Solution of thecrystal structures of CAP (Weber and Steitz, 1987) and a recombinantbovine PKA R1α subunit (Su, et al., 1995) has demonstrated that theircyclic nucleotide binding sites are formed from an a helix (A helix), aneight stranded β-roll, and two more α-helices (B and C), with theC-helix forming the back of the binding pocket. Of the approximately 120amino acids that comprise one of these cyclic nucleotide binding sites,six are invariant in all CAP, PKA, PKG and cyclic nucleotide gatedchannels. Thus, it has been suggested that the invariant residues playimportant—and conserved—roles in the folding and/or function of the CNBsites of these diverse proteins (Shabb and Corbin, 1992; Zagotta andSiegelbaum, 1996; Weber and Steitz, 1987; Su, et al., 1995; Kumar andWeber, 1992; Scott, et al., 1996). Indeed, the crystal structure of CAP(Weber and Steitz, 1987) and the regulatory subunit of recombinantbovine Rla (Su, et al., 1995) reveals that the glycines are at turnswithin the β-roll while the glutamate and the arginine form bonds withthe ribose-phosphate of the nucleotide.

[0207] Interestingly, only three of these residues—two glycines and thearginine—appear to be conserved among the more distantly relatedvoltage-gated channels which bear the CNB site motif but whose gatingmay NOT be modulated significantly by direct binding of cyclicnucleotide (KAT1 (Hoshi, 1995) and drosophila FAG (dEAG) (Bruggeman, etal., 1993) (see FIG. 11).

[0208] Thus in the plant channel, KAT1, the first glycine is mutated toan asparagine and the alanine is changed to a threonine. In dEAG theglutamate in changed to an aspartate. Furthermore, the alignment of DEAGto the highly conserved RXA sequence in β-7 is uncertain. Often, the SAAsequence within the dEAG β-7 is aligned with the RXA consensus sequencewhich suggests that the arginine is lost and replaced with a serine. RALis aligned with the RXA consensus sequence which would indicate that thearginine is retained but the alanine is replaced with a leucine.Regardless of which alignment is considered, it is clear that thebinding site sequence of KAT1, dEAG and related channels all showdeviations from the consensus motif for a functional cyclic nucleotidebinding site. In keeping with this structural divergence, there is onlyone report that any cloned EAG is being sensitive to direct cyclicnucleotide binding (Bruggermann et al., 1993). However, this result hasnot been confirmed and it is now thought to be an artifact. There issome evidence that the gating of the plant channel KAT1, may be weaklysensitive to cyclic nucleotide modulation (Hoshi, 1995).

[0209]FIG. 13 shows a schematic representation of the cyclic nucleotidebinding site of bRET1 showing the critical interactions between thebinding site and the cyclic nucleotide.

[0210] Recent evidence has demonstrated that the third (or C) α-helixmoves relative to the agonist upon channel activation, formingadditional favorable contacts with the purine ring.

[0211] Indeed, the selective activation of bRET1 by cGMP relative toCAMP is largely determined by a residue in the C α-helix, D604 (Varnumet al., 1995). This acidic residue is thought to form two hydrogen bondswith the hydrogens on a ring nitrogen and amino group of the cGMP purinering. Unlike the cGMP selective bRET1 channel, cyclic nucleotide-gatedchannels that are activated equally well by cAMP or cGMP (fOLF1,Goulding et al., 1992; Goulding et al., 1994) or which favor activationby cAMP (rOLF2 coexpressed with rOLF1, Liman & Buck, 1994; Bradley etal., 1994) do not have an acidic residue here, but rather, have polar orhydrophobic amino acids (see Varnum, et al., 1995). Neutralization ofD604 results in a loss of the ability to form the favorable hydrogenbonds with cGMP and the loss of the unfavorable interaction with thelone pair of electrons on the purine ring of CAMP, thus accounting forthe channels which bear a hydrophobic or polar residue at this positionbecoming non-selective between CAMP and cGMP or even selective for CAMP.

[0212] In the C-terminus of the BCNG proteins there is a sequence ofapproximately 120 amino acids that is homologous to these cyclicnucleotide binding sites. Strikingly, the six residues that have beenshown to be totally conserved in all functional cyclic nucleotidebinding sites are conserved in all of the BCNG proteins that identified.The cyclic nucleotide binding site of the BCNG channels are most similarto the functional site present in the voltage-independent cyclicnucleotide-gated channels (30%). When the cyclic nucleotide bindingsites found in channel genes are compared to those in protein kinases,the BCNG channel sites are more similar (25% similarity to yeastCAMP-dependent protein kinases) than those of any other ion channel.These data strongly suggest that the BCNG genes encode proteins whoseactivity is modulated by direct binding of cyclic nucleotide.Furthermore, the BCNG channels all have an isoleucine residue in theposition where D604 is found in the cGMP selective bRET1 channel. Thus,the BCNG are suggested to be cAMP selective.

[0213] The pore. Despite the functional divergence that has given riseto Na, Ca or K selective families and to the presence of channels withinthese families whose conductances vary by 1-2 orders of magnitude, thepores of all members of the voltage-gated superfamily are related (seeItillic 1992; For example see FIG. 14). Much is known about the residuesthat contribute to the ion permeation properties of channels and thisallows predictions about the permeation properties of the BCNG proteins(Mackinnon, 1991; Heginbothem et al., 1994).

[0214] Overall, the P region of mBCNG-1 is most closely related to thecorresponding region in the K selective Shaker and eag channels, albeitpoorly (30%). Based on the presence of a GYG motif in the P loop, theBCNG proteins would be expected to be K selective. However, the BCNG Ploops contain substitutions in several positions that are otherwisehighly conserved in other voltage activated K+ channels. These changescan be seen in FIG. 14 (which shows an alignment of mBCNG-1 againstchannels from all the other major K channel families) and FIG. 8 (whichshows the alignment of all currently cloned BCNG sequences). Theaspartate residue which follows the GYG triplet is replaced with alanine(position 352) in mouse and human BCNG-1 and by an arginine in the otherBCNG channels identified so far. The serine/threonine residue 8 residuesN-terminal from that position (residue 344 in mBCNG-1) is replaced withhistidine in all of the BCNG sequences. 6 residues N-terminal from theaspartate a hydrophobic leucine residue is introduced in place of apolar residue. In addition, at position −12 from the aspartate, a sitethat is occupied by an aromatic residue in all of the other channelsaligned in FIG. 14, a lysine residue is introduced in all of the BCNGsequences (FIG. 8 and FIG. 14).

[0215] Although the amino acid substitutions seen in the P region of theBCNG subunits do not necessarily indicate that the channel will havelost its K selectivity (for example a lysine is present in the P regionof the K selective Shaw channel, See FIG. 14) the substantial deviationsfrom the K channel consensus sequence suggest that the BCNG proteins maygenerate a family of channels that do not select well between Na andK—consistent with the hypothesis that the BCNG channels encode thenon-selective Ih pacemaker current.

[0216] Discussion

[0217] Significance of the BCNG Structure. The presence of cyclicnucleotide binding sites on a number of K⁺ channels that are found inboth plant and animal phyla suggests that the fusion between anancestral K⁺ channel and an ancestral cyclic nucleotide binding site islikely to have occurred prior to the evolutionary separation betweenplants and animals (Warmke and Ganetzky, 1994). Indeed, the finding thatmany of these sites are degenerate and non-functional supports thisinterpretation. Divergence from this common ancestor would have led onone hand to Eag-related channels (EAG, ERG, ELK) (Warmke and Ganetsky,1994) and plant inward rectifiers (AKT and KAT), which maintained moreof the features of voltage activated K⁺ channels, while showing aprogressive deviation from the original cyclic nucleotide binding sitesequence) and on the other hand to CNG-channels (which show a higherevolutionary constraint on the cyclic nucleotide binding site, whilethey have lost voltage activation and K⁺ selectivity) (Anderson, et al.,1992; Sentenac, et al., 1992). The features of BCNG-1 suggest that itmay have remained closer to the ancestral molecule that represents theevolutionary link between voltage-gated K⁺ channels and cyclicnucleotide-gated channels. This is supported by the observations thatthe cyclic nucleotide binding site of mBCNG-1 shows the closest homologyto binding sites present in protein kinases, in particular in yeastcAMP-dependent protein kinases (25%) while the channel domains are mostclosely related to the voltage-dependent channel encoded by the Shakergene that does not have a cyclic nucleotide binding site and thus,presumably arose before the gene fusion event. The cyclic nucleotidebinding site of mBCNG-1 is most homologous to the site present in cyclicnucleotide-gated channels (30%) which again demonstrates that theseprobably arose from a common ancestor and in both there was pressure tomaintain the cyclic nucleotide binding site because it contributed tothe function of the protein. Thus, BCNG-1 appears to constitute a newbranch of the K⁺ channel superfamily.

[0218] Physiological Significance. Ion channels are central componentsunderlying the electrical activity of all excitable cells and serveimportant transport functions in non-excitable cells. Members of thenovel BCNG family of ion channels are expressed in both brain andcardiac muscle as well as skeletal muscle, lung, liver, pancrease andkidney. From their amino acid sequence, members of the BCNG channel genefamily are likely to have important, novel roles in theelectrophysiological activity of the brain and the heart and othertissues. This view is based, first, on the finding that mRNA coding forBCNG channel protein is expressed in both heart and brain. Second, thededuced primary amino acid sequence of the BCNG channels indicate thatthey are members of the voltage-gated channel family but unlike mostvoltage-gated channels, the BCNG channels contain what appears to be afunctional cyclic nucleotide-binding domain in their carboxy terminus.

[0219] Northern blots of the four mouse BCNG channel genes showinteresting differences in expression patterns (see FIG. 3, FIG. 9 andFIG. 10). mBCNG-1 is selectively expressed in brain. Western blotsconfirm that mBCNG-1 is also highly expressed at the protein level, andthat this expression is widespread throughout the mouse brain (see FIG.2). mBCNG-2 is expressed in brain and heart. mBCNG-3 is expressed inbrain, heart, lung and skeletal muscle. mBCNG-4 is expressed in brain,liver and kidney. Thus, each gene, although highly similar at the aminoacid level, shows a distinct pattern of expression, implying that eachhas a unique physiological function. This is borne out by the findingthat the two human members of the BCNG family, hBCNG-1 and hBCNG-2, showsimilar patterns of expression to the mouse homologs. Thus, hBCNG-1 isselectively expressed in brain (with weaker hybridization in pancrease)whereas hBCNG-2 is expressed in brain and heart. Even within aparticular organ system, the different genes show different patterns ofexpression. Thus, in the brain hBCNG-1 is more highly expressed inhippocampus and amygdala than in other brain regions. In contrast,hBCNG-2 is highly expressed in all brain regions.

[0220] Based on the BCNG amino acid sequence and tissue distribution, itwas hypothesized that the channels encode a either a voltage-gatedpotassium channel that is activated by membrane depolarization andmodulated by the direct binding of cyclic nucleotides, or thehyperpolarization-activated pacemaker channel that underlies spontaneouselectrical activity in the heart (DiFrancesco and Torta, 1991) and invarious regions of the brain (Steride et al., 1993). This latterhypothesis is based on the finding that the pacemaker channels, similarto BCNG genes, are expressed in both brain and the heart. Moreover, thepacemaker channels are known to be non-selective cation channels thatare gated by both voltage and the direct binding of cyclic nucleotidesto a cytoplasmic site on the channel (DiFrancesco and Torta, 1991;Pedarzani and Storm, 1995 Larkman And Kelly, 1997, McCormick and Page,1990). To date, there is no biochemical or molecular biologicalinformation as to the nature of the pacemaker channel.

[0221] However, the similarity in tissue distribution and proposedgating mechanisms between the pacemaker channels and the BCNG channelssuggested that the BCNG genes code for one or more subunits thatcomprise the pacemaker channels.

[0222] Pacemaker channels have been studied at the electrophysiologicallevel in both cardiac tissue and central neurons. In both instances, thechannels are activated when the cell membrane voltage is made morenegative than −40 mV. These non-selective channels are permeable to bothNa and K⁺ ions. However, at the negative membrane potential range overwhich these channels open, their main effect is to allow positivelycharged sodium to enter the cell from the extracellular environment,causing the cell membrane to become more positive. This eventuallycauses the membrane voltage to reach threshold and the cell fires anaction potential. (See FIG. 15). Cyclic AMP (cAMP) is known to shift therelation between membrane voltage and channel activation, causing thechannels to turn on more rapidly when the membrane is depolarized. Thisincreases the rate of the pacemaker depolarization, increasing the rateof spontaneous action potential firing. It is this effect that underliesthe ability of epinephrine (adrenaline) to cause the heart to beatfaster. The effects of cAMP on the pacemaker current appear to occurthrough two separate molecular mechanisms. First, cAMP activates theenzyme, cAMP-dependent protein kinase (PKA), leading to an increase inlevels of protein phosphorylation. Second, CAMP is thought to directlybind to a cytoplasmic region of the pacemaker channel, producing aneffect similar to that seen with protein phosphorylation. Such directactions of CAMP have been reported both in the heart and in brain(DiFrancesco and Torta, 1991; Pedarzani and Storm, 1995).

[0223] An alternative function for the BCNG channels, that they encodefor a novel voltage-gated and cyclic nucleotide-gated potassium channel,is suggested by the amino acid region that is known to line theion-conducting pore and hence determine the ionic selectivity of thechannel. This S5-S6 loop contains a three amino acid motif, GYG, that isconserved in almost all voltage-gated K⁺ channels Heginbotham et al.,1994. The BCNG channel S5-S6 loop shows amino acid similarity with thatof other potassium channels, including the GYG motif. This suggests thatthe BCNG channels may be K⁺ selective. However, there are a number ofstriking differences in the sequence between BCNG and other K⁺ channelsthat may indicate that the BCNG channels are less K-selective comparedto other K⁺ channels, consistent with the view that the BCNG channelscode for the non-selective cation pacemaker channels that are permeableto both Na and K⁺.

[0224] The presence of mBCNG-1 in the dendrites of hippocampal pyramidalcells is particularly intriguing, as cAMP has been shown to be importantfor the establishment of some forms of long-term synaptic potentiationin these cells (Frey, et al., 1993; Boshakov, et al., 1997; Huang andKandel, 1994; Thomas, et al., 1996). The structural features of mBCNG-1predict a K⁺ conducting activity, directly modulated by cyclicnucleotide binding.

[0225] Interestingly, a current with similar characteristics has beendescribed in the hippocampal pyramidal neurons of area CA1 (Warmke, etal., 1991) where mBCNG-1 is highly expressed. This current (IQ) isbelieved to contribute to the noradrenergic modulation of hippocampalactivity, by regulating neuronal excitability in response to cAMPlevels. BCNG-1 may participate in the formation of the channelsresponsible for this type of current.

[0226] Based on the widespread tissue distribution and likely importantphysiological role of the BCNG channels in electrical signaling, drugsthat interact with these channels are of potential therapeutic use in anumber of neurological, psychiatric and cardiac diseases as well assystemic diseases of tissues such as skeletal muscle, liver and kidney.

[0227] Neurological disease: Based on the high expression of thesechannels in the hippocampus and potential role in spontaneous pacemakeractivity, they may be useful, novel targets for treatment of epilepsy.For example, by blocking these channels it may be possible to prevent ordiminish the severity of seizures. In diseases associated withhippocampal neuronal loss, such as age-related memory deficit,stroke-induced memory loss, and Alzheimers disease, a drug whichenhanced pacemaker channel activity may be of therapeutic use byincreasing neuronal activity in the hippocampus. As these channels arealso expressed in the basal ganglia and striatum, they may be potentialtargets in Parkinson's and Huntington's disease. The BCNG channels arealso highly expressed in the thalamus, where pacemaker channels havebeen shown to be important in generating spontaneous action potentialsimportant for arousal. Targeting of such channels may help treatattention deficit disorder.

[0228] Psychiatric disease: Given the high levels of expression ofhBCNG-1 in the amygdala, these channels may be targets for drugsinvolving various affective disorders and anxiety. Their high expressionin the limbic system suggests that they may also be of potential benefitin treatment of schizophrenia.

[0229] Cardiac disease: The expression of the BCNG-2 channels in theheart suggests that they may be useful targets for treatment of certaincardiac arrhythmias. Based on the hypothesis that these genes may encodepacemaker channels, the BCNG channels will be potential targets fortreating both bradyarrhythmias through drugs that enhance pacemakerchannel activity and certain tachyarrhythmias due to enhancedautomaticity. Even if the BCNG channels are not the pacemaker channels,they are likely to play an important role in cardiac electricalactivity, perhaps contributing to action potential repolarization, andthus would remain attractive targets for drug development.

[0230] A number of drugs, toxins and endogenous compounds are known tointeract with various types of ion channels. These drugs have proveduseful as local anesthetics and in the treatment of cardiac arrhythmias,hypertension, epilepsy and anxiety.

[0231] These drugs fall into several classes including pore blockers,allosteric modulators, and competitive antagonists (see Table II). TheBCNG channels present some unique features that make them veryattractive drugs. First, there are both brain specific genes (BCNG-1)and genes that expressed in both brain and heart (BCNG-2,3). Thus BCNGmay yield drugs specific for brain or heart. Second, the pore region ofthe BCNG channels shows considerable divergence from that of other knownpotassium channels. Thus, yielding pore-blocking drugs that wouldselectively alter BCNG channels but spare other types of voltage-gatedK⁺ channels. Third, the cyclic nucleotide-binding site elucidate anotherimportant target with respect to the opening of the BCNG channels. Bydesigning specific cyclic nucleotide analogs it should be possible todesign either synthetic agonists, which will increase channel opening,or antagonists which will decrease channel opening. Most available drugsfor ion channels decrease channel opening, relatively few increasechannel opening. The ability to either increase or decrease the openingof BCNG channels offers much potential for therapeutically effectivecompounds. For example in bovine photoreceptor CNG channels, Rp-cGMPS isan antagonist of channel opening whereas Sp-cGMPS is an agonist (Kramerand Tibbs, 1996). Moreover, the amino acid sequence of the cyclicnucleotide binding site of the BCNG channels shows considerabledivergence with cyclic nucleotide binding sites of protein kinases andthe cyclic nucleotide-gated channels of olfactory and photoreceptorneurons. Thus it should be possible to design cyclic nucleotide analogswhich specifically target the BCNG channels. TABLE II Selected Drugs andToxins That Interact with Members of the Voltage-gated Ion ChannelFamily Channel Site of Therapeutic Compound Targets action Uses LocalAnesthetics Na Pore (56) Local (lidocaine, analgesia, procaine, etc.)arrhythmias diphenylhydantoin Na Pore Seizures Tetrodotoxin Na PoreSaxitoxin Na Pore α, β-Scorpion Na Activation toxin and Inactivationgates Dihydropyridines Ca (L-type) Pore arrhythmias, hypertension,angina Verapamil Ca (L-type) Pore Diltiazem Ca (L-type) Pore w-conotoxinCa (N-type) Pore w-agatoxin Ca (P-type) Pore Tetraethylammonium K Pore4-aminopyridine K Pore charybdotoxin K Pore hanatoxin K activation Gateamiodarone K ? arrhythmias 1-cis-diltiazem CNG Pore Rp-cAMPS CNG Bindingsite antagonist Sp-cAMPS CNG Binding site agonist

[0232] From their amino acid sequence, these channels are likely to havethree important physiological properties that make them a prioriattractive targets for drug development. First, their gating should bevoltage-dependent and thus it should be sensitive to modulation of thevoltage-gating mechanism. Second, they possess a cyclic nucleotidebinding domain in their C-terminus and it is probable that their gatingwill be modulated by direct binding of cyclic nucleotides. Third, theunusual sequence of the pore forming domain of the BCNG channels shouldallow the ion conduction properties of the channel to be selectivelytargeted.

[0233] If, the gating of the channels involves both the voltage-sensormachinery and the cyclic nucleotide binding site it is likely thatcoordinated drug regimes such that two compounds with low efficacy andeven low selectivity can combine to selectively target the BCNGchannels. Thus one compound that alone would have weak pharmacologicaleffects on many voltage-activated channels combined with one that has asimilarly weak effect on the various cyclic nucleotide binding pocketscould be applied together. As no class of molecules is currently knownthat functionally combines BOTH of these structural elements—with theanticipated exception of the BCNG channels—it is likely that such aregime would lead to a highly efficacious and selective targeting ofchannels containing the BCNG subunits. Selective intervention againstBCNG sub-types should also be possible.

[0234] The regulation of these channels through drugs provides a uniqueopportunity for regulating electrical activity associated with diseasesas diverse as epilepsy and cardiac arrhythmias. Moreover, the cyclicnucleotide binding domain of these channels provides a uniquepharmacological target that could be used to develop novel, specific,cyclic nucleotide agonists or antagonists to upregulate or downregulatechannel function.

[0235] Drugs can modulate voltage-dependent gating-coupled with CNG toachieve selectivity.

[0236] Cell lines expressing mBCNG-1, mBCNG-2, mBCNG-3, mBCNG-4, hBCNG-1and hBCNG-2 offer the promise of rapid screening for compounds thatinteract with the channels. To identify drugs that interact with thecyclic nucleotide binding domain, this region could be expressedselectively in bacteria and then purified. The purified protein fragmentcould then be used in standard ligand-binding assays to detect cyclicnucleotide analogs that bind with high affinity.

[0237] Functional effects of drugs on channel opening or ion permeationthrough the pore are tested using whole cell patch clamp of mammaliancell lines expressing the various BCNG genes. Where the BCNG channelsresemble the CNG channels, they exhibit significant permeability tocalcium. This permits a high throughput screen in which channel functionis assessed by imaging intracellular calcium concentration. Drugs thatincrease channel opening also increase internal calcium.

EXAMPLE 4

[0238] Functional Expression of mBCNG-1 in Xenopus Oocytes Reveals aHyperpolarization-activated Cation Current Similar to Native PacemakerCurrent. [Identification of a gene encoding ahyperpolarization-activated “pacemaker” channel of brain].

[0239] Introduction

[0240] The generation of pacemaker activity in heart and brain ismediated by hyperpolarization-activated cation channels that aredirectly regulated by cyclic nucleotides. We previously cloned a novelmember of the voltage-gated K channel family from mouse brain (mBCNG-1)that contained a carboxy-terminal cyclic nucleotide-binding domain(Santoro et al., 1997) and hence proposed it to be a candidate gene forpacemaker channels. Heterologous expression of mBCNG-1 demonstrates thatit does indeed code for a channel with properties indistinguishable frompacemaker channels in brain and similar to those in heart. Threeadditional mouse genes and two human genes closely related to mBCNG-1display unique patterns of mRNA expression in different tissues,including brain and heart, demonstrating that these channels constitutea widely-expressed gene family.

[0241] The electrical activity of both the heart and the brain dependson specialized cells which act as pacemakers, generating the rhythmic,spontaneous firing of action potentials which can control muscleactivity, certain rhythmic autonomic functions, and particularbehavioral states. In normal nerve or muscle cells, pacemaker activityis characterized by spontaneous firing of action potentials that isintrinsic to the cell and independent of synaptic input. Defects inpacemaker activity can lead to both inherited (Spellberg, 1971) andacquired (Bigger and Reiffel, 1979) cardiac arrhythmias and may alsounderlie various neurological diseases.

[0242] In many of these cases, the pacemaker activity is generated by ahyperpolarization-activated channel that is permeable to both sodium andpotassium and is present in both heart (DiFrancesco, 1993) and brain(Pape, 1996). Such cation-permeable channels were initially described incardiac sinoatrial node cells (Brown et al., 1979; Yanagihara andIrisawa, 1980; Brown and DiFrancesco, 1980; DiFrancesco, 1986), wherethey were termed I_(f) or I_(h). They have since been described incardiac Purkinje fibers (DiFrancesco, 1981), ventricular muscle (Yu etal., 1993), and both peripheral (Mayer and Westbrook, 1983) and centralneurons (Halliwell and Adams, 1982; see Pape, 1996 for review), wherethey are referred to as I_(h) or I_(q). In sinoatrial node cells of theheart, the best studied example, this pacemaker channel drives therhythmic firing and beating of the atria and ventricles (Brown et al.,1979; Yanagihara and Irisawa, 1980; Brown and DiFrancesco, 1980;although see Irisawa et al., 1993). In fact., it is through themodulation of this pacemaker channel that acetylcholine andnorepinephrine exert their classical actions on heart rhythm.

[0243] In the brain, the modulation of pacemaker channel activity inthalamic relay neurons is important for regulating arousal during thesleep-wake cycle (Pape and McCormick, 1989; McCormick and Bal, 1997).Pacemaker activity in brainstem nuclei is likely to contribute torespiratory rhythms (Johnson and Getting, 1991; Dekin, 1993). Finallypacemaker activity in higher cortical regions is thought to contributeto endogenous oscillations that may be important for synchronizing theactivity of neuronal populations (Maccaferri and McBain, 1996; Strata etal., 1997), a synchronization that has been proposed to bind togetherthe separate analyzed components of a perceptual representation (Singerand Gray, 1995). Although the role of this channel in pacemaker activitymay be its best characterized action, it also contributes to reboundexcitation following hyperpolarizing responses in non-pacemaking cells(Fain et al., 1978; Attwell and Wilson, 1980; Wollmuth and Hille, 1992;Halliwell and Adams, 1982; Mayer and Westbrook, 1983) and may haveadditional functional roles.

[0244] One striking feature of the pacemaker channels is that theiractivity can be modulated by transmitters and hormones acting throughthe second messenger cAMP (Tsien, 1974; DiFrancesco and Tortorra, 1991).Elevation of cAMP levels shifts the voltage-dependence of pacemakerchannel activation by 0.2-10 mV in the positive direction. As a result,the channels activate more rapidly and more completely uponrepolarization to a fixed, negative potential. Indeed, it is the abilityof cAMP to modulate the activation of the pacemaker current that islargely responsible for the increase in heart rate in response toβ-adrenergic agonists (Brown et al., 1979) and the slowing of the heartrate during vaginal stimulation (DiFrancesco et al., 1989; Zaza et al.,0.1996). Intriguingly, this effect of cAMP appears to be mediatedthrough its direct binding to the channel in both sinoatrial node cells(DiFrancesco and Tortora, 1991) and in neurons (Pedarzani and Storm,1995; Ingram and Williams, 1996). By contrast, If is regulated byPKA-dependent protein phosphorylation in cardiac Purkinje cells (Changet al., 1991).

[0245] Pacemaker activity is characterized by spontaneous firing ofaction potentials in a nerve or muscle cell that is intrinsic to thecell and independent of synaptic input. This spontaneous firing isgenerated by a slow, pacemaker depolarization that is thought to involvethe turning on of the hyperpolarization-activated pacemaker channels(DiFrancesco, 1993). Such cation-permeable channels were initiallydescribed in cardiac sinoatrial node cells (Brown et al., 1979;Yanagihara and Irisawa, 1980; Brown and DiFrancesco, 1980; DiFrancesco,1986), where they were termed I_(f) or I_(h). They have since beendescribed in cardiac Purkinje fibers (DiFrancesco, 1981), ventricularmuscle (Yu et al., 1993), and both peripheral (Mayer and Westbrook,1983) and central neurons (Halliwell and Adams, 1982; see Pape, 1996 forreview), where they are referred to as I_(h) or I_(q).

[0246] The pacemaker channels, unlike most voltage-gated channels, areclosed when the membrane is depolarized during an action potential andonly open when the membrane repolarizes to negative voltages. Theopening of these channels upon repolarization of the action potentialpermits an influx of positively charged sodium ions, contributing to thespontaneous pacemaker depolarization. If this depolarization is ofsufficient amplitude it can then trigger a second action potential,leading to repetitive, rhythmic, electrical activity. Although there ismuch evidence that supports a role for these channels in pacemaking(DiFrancesco, 1993; 1995; Pape, 1996), their exact quantitativecontribution remains controversial (Irisawa et al., 1993; Vassalle,1995).

[0247] One striking feature of the pacemaker channels is that theiractivity can be modulated by transmitters and hormones acting throughthe second messenger cAMP (Tsien, 1974; DiFrancesco et al., 1986).Elevation of cAMP levels shifts the voltage dependence of pacemakerchannel activation by 5-10 mV in the positive direction. As a result,the channels activate more rapidly and more completely uponrepolarization to a fixed, negative potential. Indeed, it is the abilityof cAMP to speed up the activation of the pacemaker current that islargely responsible for the increase in heart rate in response toβ-adrenergic agonists (Brown et al., 1979) and the slowing of the heartrate during vagal stimulation, when ACh acts through muscarinic receptorstimulation to decrease cAMP levels (DiFrancesco et al., 1989; Zaza etal., 1996) Intriguingly, this effect of cAMP appears to be mediatedthrough its direct binding to the channel in both sinoatrial node cells(DiFrancesco and Tortora, 1991; DiFrancesco and Mangoni, 1994; Bois etal., 1997) and in neurons (Pedarzani and Storm, 1995; Ingram andWilliams, 1996). By contrast, If is regulated by PKA-dependent proteinphosphorylation in cardiac Purkinje cells (Chang et al., 1991).

[0248] Despite the intense physiological characterization of pacemakerfunction and mechanisms, the molecular nature of thehyperpolarization-activated cation channel that is responsible forgenerating the pacemaker depolarization has not yet been identified. Forseveral reasons, we suspected that one candidate gene for the pacemakerchannel might be mBCNG-1, which was originally cloned from a mouse braincDNA library based on its interaction with the SH3 domain of a neuralspecific isoform of Src (Santoro et al., 1997). First, the deduced aminoacid sequence of mBCNG-1 (originally termed BCNG-1 by Santoro et al.)reveals it to be a member of the superfamily of voltage-gated K channels(Jan and Jan, 1997), but with an unusual pore. Second, the carboxyterminus has a conserved cyclic nucleotide-binding (CNB) domain that ishomologous to CNB domains of protein kinases (Shabb and Corbin, 1992)and the cyclic nucleotide-gated channels (Zagotta and Siegelbaum, 1996).This suggests its gating may be directly regulated by cyclicnucleotides. Third, both mBCNG-1 mRNA and protein are widely expressedin brain. mBCNG-1 was a candidate gene for the pacemaker channel(Santoro et al., 1997), which codes for a member of the superfamily ofvoltage-gated K channels (Jan and Jan, 1997). This channel wasoriginally cloned from a mouse brain cDNA library based on itsinteraction with the SH3 domain of a neural specific isoform of Src(n-Src). The channel protein and mRNA are widely expressed in brain. Thededuced amino acid sequence of mBCNG-1 (Seq.ID.No.:2) (originally termedBCNG-1 by Santoro et al.) contains a carboxy terminus, cyclicnucleotide-binding domain that is homologous to CNB domains of proteinkinases (Shabb and Corbin, 1992) and the cyclic nucleotide-gatedchannels (Zagotta and Siegelbaum, 1996). Based on the fact that mBCNG-1appeared to encode for a subunit of a voltage-gated channel that couldbe directly regulated by cyclic nucleotides and its widespreaddistribution in the brain, it was suggested that mBCNG-1 might code fora subunit of the hyperpolarization-activated cation current (Santoro etal., 1997).

[0249] Here we report the functional expression of mBCNG-1 in Xenopusoocytes. Patch clamp recordings clearly demonstrate that this geneencodes a hyperpolarization-activated cation channel that is identicalin its four key properties to the endogenous pacemaker channel of brainand also bears considerable similarity to that of the heart. Moreover,we report partial cDNA clones coding for three additional members of theBCNG family from mouse brain (mBCNG-2,3,4). These three additionalclones are also expressed in a variety of other tissues, including theheart. Because of the potential clinical importance of this gene family,we have also isolated and characterized two human clones that are highlyconserved with and show similar expression patterns to mBCNG-1 andmBCNG-2. Thus, the BCNG channel genes may encode not only pacemakerchannels of the brain but they may also encode a family of channels thatare widely expressed in a variety of tissues, including the heart.

[0250] Results

[0251] The location and deduced amino acid sequence of mBCNG-1, a genewhich encodes a novel memeber of the superfamily of of voltage-gated Kchannels, suggested to us that it might encode thehperpolarization-activated pacemaker-type channel present in brain. Totest this idea directly, we synthesized mBCNG-1 cRNA, expressed it inXenopus oocytes, and analyzed the functional properties of the expressedchannels in cell-free membrane patches. The native brain pacemakerchannel has four distinctive properties: 1) it is activated with slowkinetics by hyperpolarization, 2) it is a cation channel that selectsweakly for K relative to Na; 3) it is blocked by external Cs but not byBa, and 4) it is directly modulated by intracellular cAMP. Expression ofmBCNG-1 generates channels with each of these four properties as isdemonstrated below.

[0252] Functional expression of mBCNG-1 in Xenopus oocytes reveals ahyperpolarization-activated cation current similar to native neuronalpacemaker current.

[0253] Patches obtained from oocytes injected with mBCNG-1 cRNA displayhyperpolarization-activated ionic currents that resemble those seen innative neuronal pacemakers (FIGS. 16A-F). These currents activate whenthe membrane is hyperpolarized from a holding potential of −40 mV totest potentials below −80 mV (FIGS. 16A, 16B). The inward currents turnon with a relatively slow time course at less negative potentials buttheir rate of activation speeds up with increasing levels ofhyperpolarization (FIGS. 16A, 16E, 16F). Upon return to the holdingpotential (−40 mV), the currents deactivate relatively quickly,generating a decaying, inward tail current (FIG. 16C).

[0254] Hyperpolarizing steps to −130 mV typically activate between −20pA to −200 pA of current among the >50 different patches from which wehave recorded (patches with less than 5 pA of current were frequentlyobserved but could not be analyzed). Such currents were not observed inuninjected control oocytes or in oocytes injected with cRNA encoding thebovine rod photoreceptor CNG channel, a voltage-independent channel thatis activated by cGMP. As the single channel conductance of I_(f) isaround 1 ps (DiFrancesco, 1986), we estimate that these patches (−1-2μm² membrane area) typically contain 50-1500 mBCNG-1 channels,indicating a robust level of expression. The absence of discernablesingle channel currents in patches that display low current densities(<2 pA), is consistent with mBCNG-1 channels displaying the small singlechannel conductance of the native pacemaker channels.

[0255] Both the time course of activation upon hyperpolarization andtime course of deactivation upon return to the holding potential showcharacteristic sigmoidal kinetics (FIGS. 16C, 16E), similar to thosereported for the If current in native cells (DiFrancesco, 1984).Following an initial lag, the time course of activation could beapproximated by a single exponential function (FIG. 16E), the timeconstant of which decreases from 290±37 ms at −105 mV (mean±s.e.m., n=5)to 98±14 ms (n=5) at −130 mV (FIG. 16F, Table III). TABLE IIIBiophysical Properties of mBCNG-1 2 mM CONTROL cAMP Cs V_(1/2) Slopeτ⁻¹³⁰ τ⁻¹⁰⁵ ΔV_(1/2) Slope % (mV) (mV) (ms) (ms) (mV) (mV) inhibitionMEAN −99.9 −5.96 97.8 287.4 1.8 −6.30 92.4 SEM 0.8 0.71 13.6 37.0 0.30.71 2.5 n 5 5 5 5 5 5 6

[0256] TABLE III. SUMMARY OF BIOPHYSICAL PROPERTIES OF mBCNG-1. Control.Average values for steady-state activation parameters: V_(1/2) andslope, from Boltzmann equation fit to tail-current activation curves;mean time constants of activation for steps to −130 and −105 mV. cAMP.Mean effect of cAMP on V_(1/2) and slope of steady-state activationcurve. Shift in V_(1/2) measured by averaging V_(1/2) values before cAMPand after washout and subtracting this average from V_(1/2) value inpresence of cAMP (1, 30 or 3000 μM). Slope gives mean slope in presenceof cAMP. 2 mM Cs. Mean percent block of mBCNG-1 current by Cs. Secondand third lines show standard errors and number of experiments for eachmeasurement.

[0257] The steady-state voltage-dependence of activation was determinedby hyperpolarizing the membrane to various test voltages (FIG. 16C). Therelative magnitude of the decaying inward tail current upon return tothe holding potential of −40 mV provides a measure of the fractionalactivation of the current during the preceding hyperpolarization. Thepeak tail current amplitudes show a sigmoidal dependence on the testvoltage (FIG. 16D). They begin to activate at potentials negative to −80mV and reach maximal activation with steps to between −110 and −120 mV.Fits of the Boltzmann equation to this relation (see ExperimentalProcedures) yield estimates of the voltage at which mBCNG-1 ishalf-maximally activated (V_(1/2)=−99.9±0.8 mV, n=5) as well as theslope of the relation between voltage and activation (e-fold change for−6.0±0.7 mV, n=5).

[0258] The mBCNG-1 Current is Carried by K and Na

[0259] Native pacemaker channels are weakly selective for K over Na,exhibiting typical reversal potentials of around −30 mV underphysiological gradients of Na and K. To demonstrate that the mBCNG-1current is indeed mediated by cation-conducting channels, we measuredthe reversal potential of these currents under conditions of symmetricalCl concentrations but asymmetric concentrations of K and Na thatapproximate their physiological gradients (FIGS. 17A, 17C). We find thatthe reversal potential of the mBCNG-1 current occurs at −31.8±1.6 mV(n=4), very close to the expected value (FIGS. 17A-C). As the abovemeasurements were obtained in the absence of added Ca, we next exploredthe possibility that these channels may be converted to Ca-selectivechannels in the presence of external Ca, similar to voltage-gatedcalcium channels (Hess and Tsien, 1983). However, we found that additionof 1 mM Ca to the external NaCl solution did not cause any positiveshift in the reversal potential (−34.9±3 mV, n=2), which would beexpected for a Ca-selective channel.

[0260] The reversal potential that we determined for the mBCNG-1 currentis also clearly distinct from the value of 0 mV expected for a currentcarried by chloride ions. This point is important as Xenopus oocytescontain endogenous hyperpolarization-activated Cl channels whose levelof expression can change upon expression of cRNAs (Tzounopolous et al.,1995). To further rule out a role for Cl, either as a charge carrierthrough mBCNG-1 channels or as a current that contaminates ourmeasurements of mBCNG-1 currents, we replaced the internal Cl byaspartate, an anion which does not permeate most Cl channels. This isexpected to shift the Cl equilibrium potential to −78 mV. Although Clreplacement altered the magnitude of the mBCNG-1 current (similar tothat previously reported for the native If channels—Frace et al., 1992),it did not cause a negative shift in the reversal potential (FIGS. 17B,17C; there may be a small positive voltage shift that might be due tochanges in liquid junction potential or K ion activity). It was thusconcluded that mBCNG-1 does indeed code for ahyperpolarization-activated cation channel that is permeable to K andNa, similar to native I_(f) and _(h)I currents. Based on the measuredreversal potential and the Goldman-Hodgkin-Katz equation, the channel is4-fold more permeable to K than to Na, similar to the ratio in nativepacemaker channels.

[0261] The mBCNG-1 Current is Blocked by External Cs but not by ExternalBa

[0262] A characteristic feature of the pacemaker channel that allows itto be distinguished from several other types ofhyperpolarization-activated channels is its sensitivity to block byrelatively low concentrations of extracellular Cs (DiFrancesco, 1982;Noma et al., 1983). At the same time, the pacemaker channels arerelatively insensitive to block by external Ba (DiFrancesco, 1982). Wefind that the mBCNG-1 channels are similar to native pacemaker channelsin their sensitivity to external cations. Thus, the mBCNG-1 current isnearly completely blocked by 2 mM Cs ions when applied to theextracellular surface of an outside—out patch (FIGS. 18A-18B; mean %inhibition=92.4±2.5, n=6). Dose response curves for this effect showthat the IC₅₀ is around 200 μM with a Hill coefficient of −1 (FIG. 18C).In contrast to the blocking action of Cs, addition of 1 mM Ba to theexternal solution, which blocks inward rectifier K channels andhyperpolarization activated Cl channels, had little effect (FIGS. 18A-B;mean percent inhibition=0 ±5%, n=3). The fact that mBCNG-1 channels arelargely blocked by Cs also indicates that our current measurements arenot contaminated to any significant extent by endogenous oocyte Clchannels (Barish, 1983; Tzounopolous et al., 1995) or stretch-activatedcation channels (Yang and Sachs, 1990), neither of which are blocked byexternal Cs.

[0263] mBCNG-1 Channels are Directly Modulated by cAMP

[0264] Previous studies on native pacemaker channels have shown thatdirect application of cAMP and/or cGMP to cell-free inside-out patchescan increase the size of the If current elicited by a submaximalhyperpolarization, due to a shift of the activation curve to morepositive potentials (DiFrancesco and Tortora, 1991). We observed aqualitatively similar effect with the mBCNG-1 channels (FIGS. 19A-C).Thus, in resonse to bath application of cAMP to the internal surface ofan inside-out patch, there is a reversible increase in the magnitude ofthe inward current during a step to 100 mV. The increase in current isobserved in response to 1 μM, 30 μM or 3 mM cAMP (FIGS. 19A, 19B).

[0265] This effect of CAMP is due to a small, but reproducible, positiveshift in the steady-state activation curve of these channels by 2 mV(FIG. 19C; mean=1.8+0.3 mV, n=5). Although this effect of cAMP is small,it was consistently observed in 5 out of 5 patches and found to bestatistically significant (P<0.001; paired t-test). Moreover, in thefour patches in which we could record full activation curves afterwashout of CAMP, the shift was shown to be reversible. The voltage shiftis also observed when the individual activation curves from the 5separate experiments are averaged to obtain the mean activation curvesin either the absence or presence of cAMP (FIG. 19C). Although theaveraging procedure tends to obscure the effects of cAMP due to smallvariability in the control activation curves among different patches,the difference between the curves is still significant (P<0.05; two wayANOVA with one repeated measure, F(3, 6)=11.56). Furthermore, the shiftwe observe here with cAMP is nearly identical to the shift observed forpacemaker channels in the hippocampus (Pedarzani and Storm, 1995), aregion where mBCNG-1 is highly expressed (Santoro et al., 1997). Asthere was no ATP or GTP in the internal solutions, cAMP is likely to actby directly binding to the channels.

[0266] Based upon the above observations, we concluded that thehyperpolarization-activated current observed upon mBCNG-1 expressionrepresents expression of a pacemaker channel with voltage-dependence,ionic selectivity, ionic blocking properties and second messengermodulation similar to the native brain pacemaker channels.

[0267] Identification of a Family of BCNG Genes

[0268] Since mBCNG-1 is expressed only in brain, we wondered whetherother, related genes may be expressed in different tissues, includingthe heart. We have isolated partial cDNA clones for three additionalmouse and two human genes encoding regions homologous to mBCNG-1.Partial cDNA clones representing two mouse genes (mBCNG-2 and mBCNG-3)were isolated while screening for full-length mBCNG-1 products, and afourth mouse gene (MBCNG-4) as well as two human genes. (hBCNG-1 andhBCNG-2) were identified following an EST database homology search,using the protein sequence of mBCNG-1 as a query. Further extensions ofthe identified cDNA clones were subsequently obtained by libraryscreening or RT-PCR cloning. A schematic representation of the mouse andhuman BCNG sequences identified so far is presented in FIGS. 7A-7B.

[0269] Predicted Amino Acid Sequence of the Conserved BCNG ChannelFamily

[0270] The deduced, integrated, partial sequences obtained so far forthe mBCNG-2, mBCNG-3 and mBCNG-4 encoded proteins are shown in FIGS.8A-8B, and are tentatively aligned to the previously reported fulllength sequence of mBCNG-1 (Santoro et al., 1997). All of the identifiedsequences (except mBCNG-4) contain the conserved motifs of avoltage-gated potassium channel (Jan and Jan, 1997), including the S1-S6transmembrane segments, a charged S4 voltage-sensor, and a pore-lining Ploop. In addition, all BCNG family members contain a conserved cyclicnucleotide-binding domain in their carboxy terminus. It is interestingthat both mBCNG-1 and mBCNG-2 contain a serine residue in theircytoplasmic carboxy terminus that lies within a consensus site for PKAphosphorylation (FIGS. 7a-7B, arrow) whereas mBCNG-4 does not containthis site. The absence or presence of the PKA site on different channelsmay explain why the cardiac Purkinje fiber channel is regulated by PKAphosphorylation (Chang et al., 1991) whereas the sinoatrial node channelis directly regulated by cAMP (DiFrancesco and Tortora, 1991).

[0271] The three mouse proteins are closely related to each other,having a similarity of 84-86% over the hydrophobic core region (aminoacids 111 through 419, numbered according to mBCNG-1). Notably, mBCNG-2and mBCNG-3 are more closely related to each other (89% similar) thaneither is to mBCNG-1. As far as a limited alignment could show, mBCNG-4appears to be the most distantly related protein in the group (aminoacids 529 through 592), having a similarity of 79% to mBCNG-1. The 308amino acid-long core region of the hBCNG-1 protein, extending from theS1 through the S6 transmembrane segments, is 100% identical to mBCNG-1,while the core region of the hBCNG-2 protein is 98% similar to mBCNG-2.

[0272] Tissue Distribution of BCNG mRNA Expression

[0273] Northern blot analysis showed individual patterns of tissuedistribution for each of the identified clones and a high correspondencein the transcript and localization patterns between homologous mouse andhuman clones (FIGS. 9A-9D and 10A-10D). The expression of mBCNG-1appears to be largely restricted to the brain (FIGS. 9A-D; probe “q1”,see FIGS. 7A-7B), as previously reported using a distinct amino terminusprobe (probe “q0”; Santoro et al. 1997). In contrast, mBCNG-2 andmBCNG-3 are expressed in the brain as well as in the heart (FIGS. 9A-D).Hybridization signals for mBCNG-3 are also detected in poly A+ RNA fromskeletal muscle and lung. A distinct pattern of tissue distribution isrevealed for mBCNG-4, which appears to be mainly expressed in the liver,but is also present in brain, lung and kidney (FIGS. 9A-D).

[0274] The homologous mouse and human BCNG genes are likely to befunctionally similar since they exhibit very similar patterns of tissueexpression as revealed by Northern blot analysis. FIGS. 10A-D shows thata probe designed within the hBCNG-1 sequence recognized four transcriptsin human brain polyA+ RNA, similar to that seen in the mBCNG-1 Northernblot (FIGS. 9A-9D and Santoro et al. 1997). Weak hybridization signalsare also detected for hBCNG-1 in human muscle and pancreas. Northernblot analysis using a probe based on the hBCNG-2 sequence showed anexpression pattern which is highly consistent with the expressionpattern of mBCNG-2 (FIGS. 10A-D; compare with FIGS. 9A-9D). An abundant3.4 kb transcript is detected in the brain and the same transcript isalso present in the heart.

[0275] The analysis of the distribution of mBCNG-1 within the mousebrain (Santoro et al. 1997) revealed that the highest expression ofmBCNG-1 occurs in the cortex, hippocampus and cerebellum. Northern blotanalysis of the hBCNG-1 mRNA distribution within different brain regionsalso showed a differential expression of the gene, with the highestlevels present in cortical structures (hippocampus and amygdala; FIGS.10A-D). hBCNG-2 shows a more uniform level of high expression in allbrain structures, suggesting a more ubiquitous role. In particular, thestrong hybridization signal in corpus callosum-derived RNA may indicateexpression of hBCNG-2 within glial cells.

[0276] If the BCNG-2 and BCNG-3 genes expressed in heart do indeed codefor pacemaker channels, we would expect them to be expressed in thecardiac tissues in which pacemaker channel activity has been reported.Although the sinoatrial node is the primary pacemaking tissue of theheart, latent pacemaker activity is found in both atrial (Thuringer etal., 1992; Zhou et al., 1992) and ventricular (Yu et al., 1993; Robinsonet al., 1997) muscle. However, the voltage-dependence of activation ofthe channels in the regions outside of the node is normally shifted tovery hyperpolarized, non-physiological potentials. In accord with thisobservation of a widespread occurrence of pacemaker channels throughoutthe heart, we find that primers which amplify both mBCNG-2 and mBCNG-3,but not mBCNG-1, generate RT-PCR products from ventricular, atrial andsinoatrial node mRNA (FIG. 20A). To establish the relative expression ofmBCNG-2 and mBCNG-3 within each of these regions of the heart, weperformed a Southern blot analysis on the RT-PCR products. Hybridizationwith probes that specifically recognize mBCNG-2 or mBCNG-3 demonstratethat the predominant species within the RT-PCR product, and hence theanalyzed cardiac regions, is mBCNG-2 (FIGS. 20B, C).

[0277] Discussion

[0278] Identification of a Pacemaker Channel Gene for Brain

[0279] The distinct sequences and tissue distributions of the identifiedBCNG genes reveals that the BCNG products represent a family of ionchannel proteins, with characteristic motifs for voltage-sensing andcyclic nucleotide-binding. These genes are predominantly located inbrain and in heart. When expressed in Xenopus oocytes, the mBCNG-1channel gives rise to a hyperpolarization-activated cation channel whoseproperties closely correspond to those of the pacemaker current in theheart (I_(f)). Although we have no direct evidence that the members ofthe BCNG channel family expressed in heart code for cardiac pacemakerchannels, the tissue distribution and sequence similarity of thesepartial cDNA clones with full-length mBCNG-1 is suggestive that they maywell code for the cardiac channel.

[0280] Members of the voltage-gated K channel family are generallytetramers, composed of four pore-forming subunits (MacKinnon, 1991).Although mBCNG-1 cRNA leads to expression of functional channels on itsown, the existence of multiple BCNG genes suggests that the nativechannels may be heteromultimers. In addition, the BCNG channel subunitsmay associate with auxiliary, non-pore-forming β subunits that modifythe function of the channel. It is possible that mBCNG-1 may be morepotently modulated by cAMP when it combines with additional subunits.Alternatively, there may be inherent differences in the efficacy withwhich cAMP. modulates different BCNG family members. In support of thislatter possibility, the cAMP-dependent shift of Ih in hippocampalneurons, where BCNG-1 is prominently expressed (FIGS. 10A-D, and Santoroet al., 1997), is only 2-3 mV (Pedarzani and Storm, 1995), almostidentical to the shift observed here. This contrasts with the 10 mVshift reported for If in sin θ-atrial node (DiFrancesco and Tortora,1991) and the 4-6 mV shift of Ih in sensory neurons (Ingram andWilliams, 1966). The relatively rapid activation kinetics of mBCNG-1also make it more simlar to the rapidly activated hippocampal channel(Halliwell and Adams, 1982; Maccaferri et al., 1993; Pedarzani andStorm, 1995) than to the more slowly activating cardiac channels. It isan intriguing possibility that differences in cAMP efficacy may berelated to the phosphorylation state of the serinve residue in the PKAconsensus site within the cyclic nucleotide binding domain of the BCNG-1and BCNG-2 subunits.

[0281] Two Important Differences from Classic Voltage-Gated ChannelsEndow the mBCNG-1 Channel with its Characteristic Properties

[0282] Despite the sequence similarity between the BCNG channels andother voltage-gated K channels, there are two important functionaldifferences. First, most voltage-gated K channels are usually activatedby depolarization.

[0283] The opposite voltage-dependent polarity of the mBCNG-1 channelsoccurs despite the presence of a highly charged, basic S4voltage-sensing domain. Second, whereas most members of the K channelfamily are at least 100-fold selective for K ions over Na ions, themBCNG-1 channels, like the native pacemaker channels, are only four foldselective for potassium versus sodium. The BCNG channels do, however,contain a P region that is similar to P regions of other K selectivechannels. We discuss possible mechanisms for these differences below.

[0284] How can the BCNG channels activate upon membranehyperpolarization rather than depolarization, despite the presence of anS4 segment? A similar reversed polarity of voltage-dependent activationhas been reported for the S4-containing KAT1 voltage-gated potassiumchannel of plants (Schachtman et al., 1994). One relatively simplemechanism that can explain the voltage-gating properties of KAT1 andmBCNG-1 channels is provided by the study of Miller and Aldrich (1996)on Shaker K channels, which normally activate rapidly and theninactivate rapidly upon depolarization. These authors showed that S4point mutations that shift the activation gating reaction to verynegative potentials (well below the resting potential) transformed theShaker K channels into hyperpolarization-activated channels. At voltagesnear the resting potential, even though the activation gates of themutant channels are in the open configuration, the channels are closeddue to the inactivation reaction. Moderate hyperpolarizations, that arenot sufficiently negative to shut the activation gates, open thechannels by causing inactivation gates to open. ‘The opening ’ of theBCNG channels upon hyperpolarization could reflect a similar removal ofinactivation.

[0285] The lower K selectivity of the mBCNG-1 channels could reflect thepresence of several non-conservative changes at key positions in the Pregion. Thus, although the channels do contain the GYG motif that formsthe major part of the K channel selectivity sequence, a conservedthreonine residue two residues N terminal to the GYG triplet is changedto a cysteine in the BCNG channels. This conserved threonine has beenshown to form part of the K selectivity filter in the X-ray crystalstructure of the bacterial kcsa channel (Doyle et al., 1998). Moroever,a highly conserved aspartate (immediately C terminal to the GYG triplet)is either an alanine or arginine in the BCNG clones.

[0286] The Role of the Pacemaker Current in Disease

[0287] Might changes in BCNG channel function due to posttranslationalmodification, changes in gene expression, or genetic mutations underlieinherited or acquired neurological disorders or diseases ofautomaticity? With our identification that the BCNG genes code for CNSand, perhaps, cardiac pacemaker channels, it should now be possible todetermine whether certain familial sinus rhythm diseases (Spellberg,1971) are due to primary defects in the pacemaker channel. Defects inpacemaker channel function could also contribute to acquired diseases ofthe heart, such as sick sinus syndrome associated with atrialfibrillation, sinus tachycardias and bradycardias (Bigger and Reiffel,1979), and ventricular arrhythmias associated with heart failure (Cerbaiet al., 1994; 1997).

[0288] The existence of multiple genes coding for regionally specificchannels offers the intriguing possibility of developing therapeuticagents that would specifically target, for example, cardiac rather thanbrain pacemaker channels. Conversely, the importance of pacemakeractivity in the brain for arousal and perhaps perceptual awareness mightmake these brain channels interesting targets for pharmacologicalmanipulation. Finally, controversies as to the precise role of thepacemaker channels in the electrical activity of both the brain andheart should now be amenable to the powerful approaches of mousegenetics.

[0289] Experimental Procedures

[0290] Library Screening and RT-PCR Cloning

[0291] Standard manipulations of Escherichia coli, lambda phage andnucleic acids, including recombinant DNA procedures, were performedessentially as described (Sambrook et al. 1989).

[0292] The first fragment of mBCNG-2 cDNA was cloned as a product of PCRreactions designed to isolate full length mBCNG-1 cDNA (Santoro et al.,1997). This fragment corresponded to 234-430 of the mBCNG-2 sequence(numbering here and througout according to mBCNG-1, see FIGS. 8A-8B) andwas used to screen a mouse brain Xgt10 library (CLONTECH® ML 3000a) athight stringency, yielding the N-terminal part of mBCNG-2 (clone 11-γ1)From the same γgt10 library screen, a weakly reacting plaque was alsoidentified (clone 15-7), which was subsequently shown to represent athird distinct gene (mBCNG-3)

[0293] A BLAST search in mouse and human EST databases revealed four ESTclones that appeared to be fragments of two mouse BCNG genes (M41-EST,gb AA023393; M28-EST, gb AA238712) and two human BCNG genes (H57-EST, gbH45591; H61-EST, gb N72770). Oligonucleotides were designed within thesesequences and used together with oligonucleotides designed in conservedregions of the BCNG clones to obtain RT-PCR products from mouse or humanpolyA+ RNA (see below). RT-PCR products were sequenced and theoverlapping regions compared, to establish the correspondence betweenESTs and known BCNG clones (see FIG. 5). The M28-EST clone appeared torepresent a fourth and distinct gene from the previously identified BCNGcDNAs. Complete sequencing of the M28-EST clone revealed that only the3′ end of the clone aligns with the BCNG sequences; the sequence 5′ toposition 632 is likely to represent an intron, and a stop codon ispresent at position 869.

[0294] A human brain γgt10 library (CLONTECH®, HL 3022a) was screenedwith a fragment derived from H61-EST clone (probe “H61”); see below, andFIGS. 7A-C), yielding the N-terminal region of the hBCNG-2 sequence upto amino acid at position number 587.

[0295] RT-PCR reactions were performed (25 times for 45 sec at 94° C.,30 sec at 55° C., and 2 min at 72° C.) on polyA+ RNA preparations frommouse brain and mouse heart (CLONTECH®, 6616-1 and 6611-1), using theSuperScript Preamplification System (GIBCO-BRL®) with the followingoligonucleotides: B123 5′CAGTGGGAAGAGATTTTCCACATGACC3′ (Seq I.D. No.______) (corresponding to aa 269-277) and 41REV5′GATCATGCTGAACCTTGTGCAGCAAG3′ (Seq. I.D. No. ______) (corresponding toaa 590-5,98) or 28REV 5′CACCKCRTTGAAGTGGTCCACGCT3′ (Seq I.D. No. ______)(corresponding to aa 554-561).

[0296] For human genes, reactions were performed (25 times for 45 sec at94° C., 20 sec at 58° C., and 3 min at 72° C.) on polyA+ RNA from humanbrain and human heart (CLONTECH®, 6516-1 and 6533-1) using the followingoligonucleotides: MB1-3 5′ATGTTCGGSAGCCAGAAGGCGGTGGAG3′ (Seq I.D. No.______) (corresponding to aa 102-110) and H57.C5′CAGCTCGAACACTGGCAGTACGAC3′ (Seq I.D. No. ______) (corresponding to aa537-544).

[0297] For heart region localization of BCNG-2/3 transcripts, reactionswere performed on polyA+ RNA isolated from ventricles, atria orsinoatrial nodes of rabbit heart, using oligonucleotides: BCNG-2/F25′GAGCAGGAGCGCGTCAAGTCGGCG3′ (Seq I.D. No. ______) (corresponding to aa112-119) and BCNG-2/R2 5′GAAGATGTAGTCCACGGGGATGGA3′ (Seq I.D. No.______) (corresponding to aa 218-225).

[0298] To determine the specificity of recognition among differentfamily members, the same oligonucleotides were used on plasmid DNAs thatencoded mBCNG-1, 2 or 3 cDNAs (see Legend to FIG. 9A). These plasmidswere labeled by random priming (STRATAGENE®) and used as the probes forthe Southern blot analysis.

[0299] Northern Blots

[0300] For mouse gene expression studies, a mouse multiple tissueNorthern blot (CLONTECH®, 7762-1) was probed with PCR productsrepresenting the following regions of the mBCNG clones (see schematicrepresentation in FIGS. 7A-7B and amino acid numbering in FIGS. 8A-8B):

[0301] mBCNG-1: probe “q1” (corresponding to aa 594-720; Santoro et al.,1997);

[0302] mBCNG-2: probe “dA” (corresponding to aa 234-430).

[0303] mBCNG-3: probe “15-7” (corresponding to the mBCNG-3 sequence fromstart up to position 319).

[0304] mBCNG-4: probe “M28” (corresponding to aa 529-607 of the mBCNG-4sequence plus 180 nt of the mBCNG-4 3′ UTR; this probe was obtained as agel-purified EcoRI/BglII restriction fragment, 400 bp, from the EST-M28DNA).

[0305] For human gene expression studies, a human multiple tissueNorthern blot (CLONTECH®, 7760-1) or human brain multiple tissueNorthern blot (CLONTECH, 7750-1) was probed with the following PCRproducts:

[0306] hBCNG-1: probe “H57” (corresponding to aa 537-800).

[0307] hBCNG-2: probe “H61” (corresponding to aa 452-634).

[0308] Hybridizations were all performed in EXPRESSHYB® solution(CLONTECH®) for 1 hr at 68° C., as indicated in the manufacturer'sProtocol Handbook. Blots were washed for 10 min at room temperature in2×SSC/0.1% SDS, followed by two washes for 30 min at 65° C. in0.2×SSC/0.1% SDS. Filters were stripped between subsequenthybridizations by boiling for 5 min in 0.5% SDS/H₂O.

[0309] Electrophysiological Recordings

[0310] mBCNG-1 was subcloned into the pSD64TR expression vector. RNA wastranscribed from BamHI-linearized DNA using SP6 RNA polymerase (MessageMachine®, Ambion®) and injected into Xenopus oocytes prepared aspreviously described (Goulding et al., 1992).

[0311] Patch clamp recordings were made from cell-free inside-out andoutside-out patches 3-7 days after CRNA injection. Data were acquiredusing either an Axopatch 200A or 200B integrating patch clamp amplifier(Axon Instruments®, USA). The holding potential in all of theseexperiments was −40 mV. For simplicity, only abbreviated descriptions ofthe solutions used are given in the text and in Brief Description of theFigures, full descriptions are given below.

[0312] The KCl-EGTA solution contained: 10.7 mM KCl, 5 mM NaCl, 10 mmHEPES (pH 7.4, KOH), 1 mM EGTA. The NaCl-EGTA solution contained 107 mMNaCl, 5 mM KCl, 10 mM HEPES (pH 7.4, NaOH), 1 mM EGTA. The KCl/NaCl-EGTAand KCl/NaCl—CaCl₂ solutions contained: 82 mM KCl, 30 mM NaCl, 10 mMHEPES (pH 7.4, KOH) with either 1 mM EGTA or 1 mM CaCl₂, respectively.In some experiments we replaced Cl with aspartate in the followingKAspartate-EGTA solution: 107 mM K-Aspartate, 5 mM NaCl, 10 mM HEPES (pH7.4, KOH), 1 mM EGTA. Where appropriate, Na-cAMP was included in theintracellular solution by iso-osmolar replacement of NaCl, while CsCland BaCl2 were included in the indicated extracellular solutions byiso-osmotic replacement of NaCl or CaCl₂, respectively. A Ag—AgCl groundwire was connected to the bath solution by a 3 M KCl agar bridgeelectrode. The largest uncompensated junction potential was measured tobe 3.4 mV. Voltages have not been corrected to account for this offset.All recordings were obtained at room temperature (22-24° C.).

[0313] Voltage clamp protocols were applied using a P/N protocol tosubtract uncompensated linear leak and capacitance using either pClamp®software (v 6.0, AXON INSTRUMENTS®, with N=8) and a Pentium 100 mHz PCcomputer or the Pulse software (v 8.11, Heka, with N=10) and a MACINTOSHCENTRIS® 650 computer. Unless otherwise indicated, data were low passfiltered at 1.25 kHz (8 pole Bessel filter, Frequency Devices 902) anddigitized at 2.5 kHz using either a TL-1 DMA Interface (AXONINSTRUMENTS®) or an ITC-16 interface (INSTRUTECH CORP.®). Analyses weredone using pClamp (v 6.0, AXON INSTRUMENTS®), Pulse (v 8.11, Heka) orSigmaplot (v 4.0 SPSS Inc.).

[0314] The current-voltage relationship was obtained by measuringsteady-state current values at the end of 3 s voltage steps (currentaveraged between 2.5 s to 2.95 s) The steady-state activation curve wasdetermined from the amplitude of tail currents observed upon asubsequent depolarizing voltage step to −40 mV (current averaged between4-10 ms following the return to −40 mV). Current values were plottedversus the hyperpolarization step voltage and fitted with the Boltzmannequation:

I(V)=A ₁ +A ₂/{1+exp [−(V−V _(1/2))/slope]}

[0315] where A₁ is the offset, A₂ the amplitude, V is voltage in mV andV_(1/2) is the activation mid-point voltage. The data and the fittedBoltzmann function were then normalized to the maximum amplitude of thefit. Activation time constants were determined by fitting singleexponentials to the rising phase of the current after allowing for aninitial lag.

[0316] To determine reversal potentials under different ionicconditions, inside-out patches held at −40 mV were stepped to −130 mVfor 300 ms and then stepped back to a series of test potentials rangingfrom −60 to +20 mV in 5 mV increments. The peak amplitude of the tailcurrents were measured by averaging the data between 2 and 4 msfollowing the step to the test potential. The sensitivity of the mBCNG-1current to external Cs and Ba was determined using the outside-out patchclamp configuration and measuring the steady-state current values at theend of 1 s steps to −130 mV (current averaged between 800 ms to 990 ms).In both of these series of experiments, electrodes were coated withSylgard to minimize pipette capacitance, and data were filtered at 2.5kHz and sampled at 5 kHz.

[0317] Contamination from an endogenous Ca-activated Cl channel in theoocyte membrane (Barish, 1983) was minimized by including EGTA (1 mM)with no added calcium in the internal solution. Oocytes also contain astretch-activated cation channel that can be recognized by its largesingle channel conductance (60 pS) (Yang and Sachs, 1990). Patchescontaining such channel activity were not studied.

EXAMPLE 5 Co-Expression of BCNG Channel Subunits and Candidate Proteins.

[0318] BCNG proteins or BCNG-related proteins may form heteromultimericproteins. In order to delineate the functional roles of novel BCNGsubunits, novel BCNG subunits are coexpressed with BCNG subunits thathave overlapping tissue distribution. Voltage-gated K⁺ channels andcyclic nucleotide-gated channels both form heteromultimers. In somecases, the subunits can form complexes with completely distinct proteins(eg. KvLQT1 with MinK—(Barhanin, et al., 1996; HERG with MinK.

[0319] McDonald, et al., 1997); IrK6.1 and IrK6.2 with the sulphonylureareceptor—Isomoto, et al., 1996; Inagaki, et al., 1996). BCNG proteinsmay assemble with subunits such as MinKor ERG like subunits. Candidateproteins are selected on the basis of overlapping tissue distributionand likelihood based on known functional properties. For example, Kv1.2shows overlapping distribution with MBCNG-1 even at the subcellularlevel (Sheng, et al., 1994; Wang, et al., 1994).

[0320] Coexpression with polyA+ mRNA. If another protein can form afunctional heteromultimer with the BCNG channel proteins, co-expressionwith size fractionated mRNA from tissue (eg. heart, brain, muscle orkidney) where the appropriate BCNG subunit is expressed (as shown byNorthern blot analysis) should result in a unique current inelectrophysiological currents when the BCNG RNA is coinjected with themRNA from the tissue.

[0321] Alternative strategies to clone subunits that will modifyfunctional properties of the expressed BCNG channels include lowstringency homology screening of appropriate libraries using nucleotideprobes derived from BCNG genes or PCR amplification from genomic or cDNAusing degenerate oligonucleotides based on BCNG genes.

[0322] Yet another method to isolate other channel subunit proteins thatmay coassemble with identified BCNG family members is to use the yeasttwo hybrid system (Fields and Soug, 1989). This system was initiallyused to clone mBCNG-1 based on its interaction with the n-src SH3 domain(See, Example 1). Conserved cytoplasmic N- and C-terminal domains fromBCNG channel proteins are used as the ‘bait’ in the yeast two hybridsystem. N- and C-terminal fragments are subcloned in an appropriateplasmid (e.g. pEG202) (Zervos et al., 1983).

[0323] Sequences of BCNG Family Members: DNA and Predicted Amino AcidSequences of Mouse and Human BCNG Clones

[0324] Mouse Sequences

[0325] DNA and Predicted Amino Acid Sequence of mBCNG-1

[0326] These mouse sequences are the original DNA and predicted aminoacid sequences obtained and are those in GenBank Accession NumberAF028737. mBCNG-1 DNA open reading frameATGGAAGGCGGCGGCAAACCCAACTCCGCGTCCAACAGCCGCGACGATGGCAACAGCGTCTTCCCCTCCAAGGCGCCCGCGACGGGGCCGGTGGCGGCCGACAAGCGCCTGGGGACCCCGCCGAGGGGCGGCGCGGCCGGGAAGGAACATGGCAACTCCGTGTGCTTCAAGGTGGACGGCGGCGGAGGAGAGGAGCCGGCGGGCAGCTTCGAGGATGCCGAGGGGCCCCGGCGGCAGTATGGTTTCATGCAGAGGCAGTTCACCTCCATGCTGCAGCCTGGGGTCAACAAATTCTCCCTCCGCATGTTTGGGAGCCAGAAGGCGGTGGAGAAGGAGCAGGAAAGGGTTAAAACTGCAGGCTTCTGGATTATCCATCCGTACAGTGACTTCAGGTTTTATTGGGATTTAATCATGCTTATAATGATGGTTGGAAATTTGGTCATCATACCAGTTGGAATCACGTTCTTCACAGAGCAGACGACAACACCGTGGATTATTTTCAACGTGGCATCCGATACTGTTTTCCTGTTGGACTTAATCATGAATTTTAGGACTGGGACTGTCAATGAAGACAGCTCGGAAATCATCCTGGACCCTAAAGTGATCAAGATGAATTATTTAAAAAGCTGGTTTGTGGTGGACTTCATCTCATCGATCCCGGTGGATTATATCTTTCTCATTGTAGAGAAAGGGATGGACTCAGAAGTTTACAAGACAGCCAGAGCACTTCGTATCGTGAGGTTTACAAAAATTCTCAGTCTCTTGCGGTTATTACGCCTTTCAAGGTTAATCAGATACATACACCAGTGGGAAGAGATATTCCACATGACCTATGACCTCGCCAGTGCTGTGGTGAGGATCTTCAACCTCATTGGCATGATGCTGCTTCTGTGCCACTGGGATGGCTGTCTTCAGTTCCTGGTTCCCCTGCTGCAGGACTTCCCACCAGATTGCTGGGTTTCTCTGAATGAAATGGTTAATGATTCCTGGGGAAAACAATATTCCTACGCACTCTTCAAAGCTATGAGTCACATGCTGTGCATTGGTTATGGCGCCCAAGCCCCTGTCAGCATGTCTGACCTCTGGATTACCATGCTGAGCATGATTGTGGGCGCCACCTGCTACGCAATGTTTGTTGGCCATGCCACAGCTTTGATCCAGTCTTTGGACTCTTCAAGGAGGCAGTATCAAGAGAAGTATAAGCAAGTAGAGCAATACATGTCATTCCACAAGTTACCAGCTGACATGCGCCAGAAGATACATGATTACTATGAGCACCGATACCAAGGCAAGATCTTCGATGAAGAAAATATTCTCAGTGAGCTTAATGATCCTCTGAGAGAGGAAATAGTCAACTTCAACTGCCGGAAACTGGTGGCTACTATGCCTCTTTTTGCTAACGCCGATCCCAATTTCGTGACGGCCATGCTGAGCAAGCTGAGATTTGAGGTGTTCCAGGCCGGAGACTATATCATTCGAGAAGGAGCTGTGGGGAAGAAAATGTATTTCATCCAGCACGGTGTTGCTGGCGTTATCACCAAGTCCAGTAAAGAAATGAAGCTGACAGATGGCTCTTACTTCGGAGAGATATGCCTGCTGACCAAGGGCCGGCGCACTGCCAGTGTCCGAGCTGATACCTACTGTCGTCTTTACTCCCTTTCGGTGGACAATTTCAATGAGGTCTTGGAGGAATATCCAATGATGAGAAGAGCCTTTGAGACAGTTGCTATTGAGCGACTCGATCGGATAGGCAAGAAAAACTCTATTCTCCTGCAGAAGTTCCAGAAGGATCTAAACACTGGTGTTTTCAACAACCAGGAGAACGAGATCCTGAAGCAGATCGTGAAGCATGACCGAGAGATGGTACAAGCTATCCCTCCAATCAACTATCCTCAAATGACAGCCCTCAACTGCACATCTTCAACCACCACCCCAACCTCCCGCATGAGGACCCAATCTCCGCCAGTCTACACCGCAACCAGCGTGTCTCACAGCAATCTGCACTCACCCAGTCCCAGCACACAGACGCCCCAACCCTCAGCCATCCTTCACCCTGCTCCTATACCACAGCAGTCTGCAGTCCTCCTATACAGAGCCCCCTGGCCACACGAACTTTCCATTATGCCTCTCCCACTGCGTCCCAGCTGTCACTCATGCAGCAGCCTCAGCAGCAACTACCGCAGTCCCAGGTACAGCAGACTCAGACTCAGACTCAGCAGCAGCAGCAGCAACAGCAGCAGCAGCAGCAGCAGCAACAGCAACAACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCCACAGACACCTGGTAGCTCCACACCGAAAAATGAAGTGCACAAGAGCACACAAGCCCTTCATAACACCAACCTGACCAAAGAAGTCAGGCCCCTTTCCGCCTCGCAGCCTTCTCTGCCCCATGAGGTCTCCACTTTGATCTCCAGACCTCATCCCACTGTGGGCGAATCCCTGGCCTCTATCCCTCAACCCGTGGCAGCAGTCCACAGCACTGGCCTTCAGGCAGGGAGCAGGAGCACAGTGCCACAACGTGTCACCTTGTTCCGACAGATGTCCTCGGGAGCCATCCCCCCCAACCGAGGAGTGCCTCCAGCACCCCCTCCACCAGCAGCTGTGCAGAGAGAGTCTCCCTCAGTCCTAAATACAGACCCAGATGCAGAAAAACCCCGTTTTGCTTCGAATTTATGA mBCNG-1 predicted amino acid sequenceMEGGGKPNSASNSRDDGNSVFPSKAPATGPVAADKRLGTPPRGGAAGKEHGNSVCFKVDGGGGEEPAGSFEDAEGPRRQYGFMQRQFTSMLQPGVNKFSLRMFGSQKAVEKEQERVKTAGFWIIHPYSDFRFYWDLIMLIMMVGNLVIIPVGITFFTEQTTTPWIIFNVASDTVFLLDLIMNFRTGTVNEDSSEIILDPKVIKMNYLKSWFVVDFISSIPVDYIFLIVEKGMDSEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYDLASAVVRIFNLIGMMLLLCHWDGCLQFLVPLLQDFPPDCWVSLNEMVNDSWGKQYSYALFKAMSHMLCIGYGAQAPVSMSDLWITMLSMIVGATCYAMFVGHATALIQSLDSSRRQYQEKYKQVEQYMSFHKLPADMRQKIHDYYEHRYQGKIFDEENILSELNDPLREEIVNFNCRKLVATMPLFANADPNFVTAMLSKLRFEVFQPGDYIIREGAVGKKMYFIQHGVAGVITKSSKEMKLTDGSYFGEICLLTKGRRTASVRADTYCRLYSLSVDNFNEVLEEYPMMRRAFETVAIDRLDRIGKKNSILLQKFQKDLNTGVFNNQENEILKQIVKHDREMVQAIPPINYPQMTALNCTSSTTTPTSRMRTQSPPVYTATSLSHSNLHSPSPSTQTPQPSAILSPCSYTTAVCSPPIQSPLATRTFHYASPTASQLSLMQQPQQQLPQSQVQQTQTQTQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQPQTPGSSTPKNEVHKSTQALHNTNLTKEVRPLSASQPSLPHEVSTLISRPHPTVGESLASIPQPVAAVHSTGLQAGSRSTVPQRVTLFRQMSSGAIPPNRGVPPAPPPPAAVQRESPSVLNTDPD AEKPRFASNLDNA and predicted amino acid sequence of MBCNG-2 GenBank AccessionNumber AF064873. mBCNG-2 DNA sequenceAAGTTCTCCCTGCGGATGTTCGGCAGCCAGAAGGCCGTGGAGCGCGAGCAGGAACGCGTGAAGTCGGCGGGGGCCTGGATCATCCACCCCTACAGCGACTTCAGGTTCTACTGGGACTTCACCATGCTGTTGTTCATGGTGGGAAATCTCATTATCATTCCCGTGGGCATCACTTTCTTCAAGGACGAGACCACCGCGCCCTGGATCGTCTTCAACGTGGTCTCGGACACTTTCTTCCTCATGGACTTGGTGTTGAACTTCCGCACCGGCATTGTTATTGAGGACAACACGGAGATCATCCTGGACCCCGAGAAGATAAAGAAGAAGTACTTGCGTACGTGGTTCGTGGTGGACTTCGTGTCATCCATCCCGGTGGACTACATCTTCCTCATAGTGGAGAAGGGAATCGACTCCGAGGTCTACAAGACAGCGCGTGCTCTGCGCATCGTGCGCTTCACCAAGATCCTCAGTCTGCTGCGGCTGCTGCGGCTATCACGGCTCATCCGATATATCCACCAGTGGGAAGAGATTTTCCACATGACCTACGACCTGGCAAGTGCAGTGATGCGCATCTGTAACCTGATCAGCATGATGCTACTGCTCTGCCACTGGGACGGTTGCCTGCAGTTCCTGGTGCCCATGCTGCAAGACTTCCCCAGCGACTGCTGGGTGTCCATCAACAACATGGTGAACCACTCGTGGAGCGAGCTCTACTCGTTCGCGCTCTTCAAGGCCATGAGCCACATGCTGTGCATCGGCTACGGGCGGCAGGCGCCCGAGACATGACAGACATCTGGCTGACCATGCTCAGCATGATCGTAGGCGCCACCTGCTATGCCATGTTCATTGGGCACGCCACTGCGCTCATCCAGTCCCTGGATTCGTCACGGCGCCAATACCAGGAGAAGTACAAGCAAGTAGAGCAATACATGTCCTTCCACAAACTGCCCGCTGACTTCCGCCAGAAGATCCACGATTACTATGAACACCGGTACCAAGGGAAGATGTCTGATGAGGACAGCATCCTTGGGGAACTGAACGGGCCACTGCGTGAGGAGATTGTGAACTTCAACTGCCGGAAGCTGGTGGCTTCCATGCCGCTGTTTGCCAATGCAGACCCCAATTTCGTCACAGCCATGCTGACAAAGCTCAAATTTGAGGTCTTCCAGCCTGGAGATTACATCATCCGAGAGGGGACCATCGGGAAGAAGATGTACTTCATCCAGCATGGGGTGGTGAGCGTGCTCACCAAGGGCAACAAGGAGATGAAGCTGTCGGATGGCTCCTATTTCGGGGAGATCTGCTTGCTCACGAGGGGCCGGCGTACGGCCAGCGTGCGAGCTGACACCTACTGTCGCCTCTACTCACTGAGTGTGGACAATTTCAACGAGGTGCTGGAGGAATACCCCATGATGCGGCGTGCCTTTGAGACTGTGGCTATTGACCGGCTAGATCGCATAGGCAAGAAGAACTCCACCTTGCTGCACAAGGTTCAGCATG ATCTCAGCTCCmBCNG-2 predicted amino acid sequenceKFSLRMFGSQKAVEREQERVKSAGAWIIHPYSDFRFYWDFTMLLFMVGNLIIIPVGITFFKDETTAPWIVFNVVSDTFFLMDLVLNFRTGIVIEDNTEIILDPEKIKKKYLRTWFVVDFVSSIPVDYIFLIVEKGIDSEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYDLASAVMRICNLISMMLLLCHWDGCLQFLVPMLQDFPSDCWVSINNMVNHSWSELYSFALFKAMSHMLCIGYGRQAPESMTDIWLTMLSMIVGATCYAMFIGHATALIQSLDSSRRQYQEKYKQVEQYMSFHKLPADFRQKIHDYYEHRYQGKMSDEDSILGELNGPLREEIVNFNCRKLVASMPLFANADPNFVTAMLTKLKFEVFQPGDYIIREGTIGKKMYFIQHGVVSVLTKGNKEMKLSDGSYFGEICLLTRGRRTASVRADTYCRLYSLSVDNFNEVLEEYPMMRRAFETVAIDRLDRIGKKNSTLLHKVQH DLSS DNA andpredicted amino acid sequence of mBCNG-3 GenBank Accession NumberAF064874. mBCNG-3 DNA sequenceTGCGAGCAGCCCTCGGCGGACACCGCTATCAAAGTGGAGGGAGGCGCGGCCGCCATCGACCATATCCTCCCCGAGGCCGAGGTGCGCCTGGGCCAAAGCGGCTTCATGCAGCGCCAGTTCGGTGCCATGCTGCAACCTGGGGTCAACAAATTCTCCCTAAGGATGTTCGGCAGCCAGAAAGCGGTGGAGCGCGAGCAGGAGAGGGTTAAGTCAGCAGGGTTTTGGATTATCCACCCCTACAGTGACTTCAGATTTTACTGGGACCTGACGATGCTGTTGCTGATGGTGGGGAATCTGATCATCATACCCGTGGGCATCACCTTCTTCAAGGATGAGAACACCACACCCTGGATCGTCTTCAATGTGGTGTCAGACACATTCTTCCTCATTGACTTGGTCCTCAACTTCCGCACGGGGATCGTGGTGGAGGACAACACAGAAATCATCCTTGACCCGCAGAGGATCAAGATGAAGTACCTGAAAAGCTGGTTTGTGGTAGATTTCATCTCCTCCATACCTGTCGAATACATTTTCCTTATAGTGGAGACTCGCATTGACTCGGAGGTTTACAAAACCGCTAGGGCTGTGCGCATTGTCCGTTTCACTAAGATCCTCAGCCTCCTGCGCCTCTTGAGGCTTTCCCGCCTCATTCGATACATTCATCAGTGGGAAGAGATTTTCCACATGACCTATGACCTGGCCAGCGCCGTGGTACGCATCGTGAACCTCATTGGCATGATGCTTCTGCTGTGTCACTGGGATGGCTGCCTGCAGTTCCTAGTGCCCATGCTGCAGGACTTCCCCCATGACTGCTGGGTGTCCATCAATGGCATGGTGAATAACTCCTGGGGGAAGCAGTATTCCTACGCCCTCTTCAAGGCCATGAGCCACATGCTGTGCATTGGGTATGGACGGCAGGCACCCGTAGGCATGTCTGACGTCTGGCTCACCATGCTCAGCATGATCGTGGGGGCCACCTGCTATGCCATGTTCATCGGCCACGCCACTGCCCTCATCCAGTCGCTAGACTCCTCCCGGCGCCAGTACCAGGAGAAGTATAAACAGGTGGAGCAGTACATGTCTTTCCACAAGCTCCCGCCTGACACCCGACAGCGCATCCATGACTACTATGAACACCGTTACCAAGGCAAGATGTTTGATGAGGAAAGCATCCTGGGTGAGTTGAGTGAGCCACTTCGAGAGGAGATCATCAACTTTAACTGCCGAAAGCTGGTGGCATCCATGCCACTGTTTGCCAACGCAGATCCCAACTTTGTGACATCCATGCTGACCAAGTTGCGTTTCGAGGTCTTCCAGCCTGGGGATTACATCATCCGCGAAGGCACCATCGGCAAGAAGATGTACTTTATCCAGCACGGCGTGGTCAGCGTGCTCACTAAGGGCAACAAAGAGACCAGGCTGGCTGATGGCTCCTATTTTGGAGAGATCTGCTTGCTGACCCGGGGTCGGCGCACAGCCAGCGTCAGAGCGGATACTTAT TNCCGCCTCTACTCACTGMBCNG-3 predicted amino acid sequenceCEQPSADTAIKVEGGAAAIDHILPEAEVRLGQSGFMQRQFGAMLQPGVNKFSLRMFGSQKAVEREQERVKSAGFWIIHPYSDFRFYWDLTMLLLMVGNLIIIPVGITFFKDENTTPWIVFNVVSDTFFLIDLVLNFRTGIVVEDNTEIILDPQRIKMKYLKSWVFVDFISSIPVEYIFLIVETRIDSEVYKTARAVRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYDLASAVVRIVNLIGMMLLLCHWDGCLQFLVPMLQDFPHDCWVSINGMVNNSWGKQYSYALFKAMSHMLCIGYGRQAPVGMSDVWLTMLSMIVGATCYAMFlGHATALIQSLDSSRRQYQEKYKQVEQYMSFHKLPPDTRQRIHDYYEHRYQGKMFDEESILGELSEPLREEIINFNCRKLVASMPLFANADPNFVTSMLTKLRFEVFQPGDYIIREGTIGKKMYFIQHGVVSVLTKGNKETRLADGSYFGEICLLTRGRRTASVRADTY XRLYSL

[0327] DNA and Predicted Amino Acid Sequence of mBCNG-4

[0328] Reported are the complete DNA sequence of clone M28-EST, and theopen reading frame (ORF) encoded between positions 632 and 871 of thatDNA sequence. GenBank accession number AF064875. MBCNG-4 DNA sequenceTTTTTGGGTTTTAAAATTTATTTTATTTTTAAAAGCGTCTCCGGANANTCTAGTGCATGGCCAGGCTACAAGCTACTGGGCCAGCAACTCTGTAGGATTATTAATGACAAAAATGCAAGGACCCCATAGTTGATGGAAACCCAGGGATGAAGCAGGGCTGTCCCACAGACTTAGGCTTTGTGGAGCTGTCTGAAAACCCAGGCTGTGGCTTTGGAAGAAGTGCAGACAACCACTGCCCAGAGTGACTTAAGGTTCATACAACCATCCAGCCACCTAAGCACCCCTACCTTCAAGCATCTTGCCAGTCCCACTTTGTGTCTGTTTAGCCTGCTTTTCTCCTCCCAAGTTAGGAGTCGGGTACACCCTGGGACGGAGCAATAAGACTGGGGTTGGAGTTAATGTGTAAAATAACTGAAAAAAACATCTGGGGCTGGCAAACCTGTTTGTCTGGAAAACAGCCTTCCAGATGTGCAGGTATGGAAACAGACAGTGCTTAGAGCAGTAAGGGACCTTATACCAGCTAATCGTTCATTCTCCCAAGTATAAGGAGGAATCTGGGGGTGCTGGGTTAGCTGCTGCAGGCCTAATTGGGGGGTGGAATGGGAGCTCTGAGCTCTTCCCCGCTTTCGCAGAGATCTGCCTGCTGACTCGAGGTCGGAGAACAGCCAGTGTAAGGGCTGACACCTATTGTCGCCTCTACTCGCTCAGCGTGGACCACTTCAATGCGGTGCTTGAGGAGTTCCCAATGATGCGCAGGGCTTTTGAGACGGTGGCCATGGACCGGCTTCGGCGCATCGGTGAGGCCTGTTTACTCTGTCTGCTCTGGGTCCTGGCTGGGCCTCATCTCATGAGCCTAGCCCTGGTGCTTTGACACCACATCCCAGCCCACCCAGTTCCAGTCCATGCCTCCAGCAGGCTGTTAGCACTGTTGCTCACTAGACTTAGCCCTAGCGAGAAATTGCCGTGGAGTGTCTCCCCAAACCCTCATTCCCCGTGTCCTTCTGGGTACCAGTTCTTAACCTCACAATTTTTTATTGATAMBCNG-4 predicted amino acid sequenceEICLLTRGRRTASVRADTYCRLYSLSVDHFNAVLEEFPMMRRAFETVAMDRLRRIGEACLLCLLWVLAGPHLMSLALVL

[0329] Human Sequences:

[0330] DNA and Predicted Amino Acid Sequence of hBCNG-1

[0331] GenBank accession number AF064876. hBCNG-1 DNA sequenceAAGGAGCAGGAAAGGGTTAAAACTGCAGGCTTCTGGATTATCCACCCTTACAGTGATTTCAGGTTTTACTGGGATTTAATAATGCTCATAATGATGGTTGGAAATCTAGTCATCATACCAGTTGGAATCACATTCTTTACAGAGCAAACAACAACACCATGGATTATTTTCAATGTGGCATCAGATACAGTTTTCCTATTGGACCTGATCATGAATTTTAGGACTGGGACTGTCAATGAAGACAGTTCTGAAATCATCCTGGACCCCAAAGTGATCAAGATGAATTATTTAAAAAGCTGGTTTGTGGTTGACTTCATCTCATCCATCCCAGTGGATTATATCTTTCTTATTGTAGAAAAAGGAATGGATTCTGAAGTTTACAAGACAGCCAGGGCCCTTCGCATTGTGAGGTTTACAAAAATTCTCAGTCTCTTGCGTTTATTACGACTTTCAAGGTTAATTAGATACATACATCAATGGGAAGAGATATTCCACATGACATATGATCTCGCCAGTGCAGTGGTGAGAATTTTTAATCTCATCGGCATGATGCTGCTCCTGTGCCACTGGGATGGTTGTCTTCAGTTCTTAGTACCACTACTGCAGGACTTCCCACCAGATTGCTGGGTGTCTTTAAATGAAATGGTTAATGATTCTTGGGGAAAGCAGTATTCATACGCACTCTTCAAAGCTATGAGTCACATGCTGTGCATTGGGTATGGAGCCCAAGCCCCAGTCAGCATGTCTGACCTCTGGATTACCATGCTGAGCATGATCGTCGGGGCCACCTGCTATGCCATGTTTGTCGGCCATGCCACCGCTTTAATCCAGTCTCTGGATTCTTCGAGGCGGCAGTATCAAGAGAAGTATAAGCAAGTGGAACAATACATGTCATTCCATAAGTTACCAGCTGATATGCGTCAGAAGATACATGATTACTATGAACACAGATACCAAGGCAAAATCTTTGATGAGGAAAATATTCTCAATGAACTCAATGATCCTCTGAGAGAGGAGATAGTCAACTTCAACTGTCGGAAACTGGTGGCTACAATGCCTTTATTTGCTAATGCGGATCCTAATTTTGTGACTGCCATGCTGAGCAAGTTGAGATTTGAGGTGTTTCAACCTGGAGATTATATCATACGAGAAGGAGCCGTGGGTAAAAAAATGTATTTCATTCAACACGGTGTTGCTGGTGTCATTACAAAATCCAGTAAAGAAATGAAGCTGACAGATGGCTCTTACTTTGGAGAGATTTGCCTGCTGACCAAAGGACGTCGTACTGCCAGTGTTCGAGCTGATACATATTGTCGTCTTTACTCACTTTCCGTGGACAATTTCAACGAGGTCCTGGAGGAATATCCAATGATGAGGAGAGCCTTTGAGACAGTTGCCATTGACCGACTAGATCGAATAGGAAAGAAAAATTCAATTCTTCTGCAAAAGTTCCAGAAGGATCTGAACACTGGTGTTTTCAACAATCAGGAGAACGAAATCCTCAAGCAGATTGTGAAACATGACAGGGAGATGGTGCAGGCAATCGCTCCCATCAATTATCCTCAAATGACAACCCTGAATTCCACATCGTCTACTACGACCCCGACCTCCCGCATGAGGACACAATCTCCACCGGTGTACACAGCGACCAGCCTGTCTCACAGCAACCTGCACTCCCCCAGTCCCAGCACACAGACCCCCCAGCCATCAGCCATCCTGTCACCCTGCTCCTACACCACCGCGGTCTGCAGCCCTCCTGTACAGAGCCCTCTGGCCGCTCGAACTTTCCACTATGCCTCCCCCACCGCCTCCCAGCTGTCACTCATGCAACAGCAGCCGCAGCAGCAGGTACAGCAGTCCCAGCCGCCGCAGACTCAGCCACAGCAGCCGTCCCCGCAGCCACAGACACCTGGCAGCTCCACGCCGAAAAATGAAGTGCACAAGAGCACGCAGGCGCTTCACAACACCAACCTGACCCGGGAAGTCAGGCCATTTTCCGCCTGGCAGCCNTCGCTGCCCCATGAGGTGTCCATTTTGATTTCCAGACCCATCCCACTGTGGGGGAGTCCCTGGCCTCCATCCCTCAACCCGTGACGGCGGTCCCCGGAACGGGCCTTCAGGCAGGGGGCAGGAGCACTGTCCCGCAGCGCGTCACCTTTTTCCGACAGATGTNGTCGGGAGCCATCCCCCCGAACCGAGGAGTCCTTCCAGCACCCCTTCCACTTATCACACCCCATCCTAAAAAAhBCNG-1 predicted amino acid sequenceKEQERVKTAGFWIIHPYSDFRFYWDLIMLIMMVGNLVIIPVGITFFTEQTTTPWIIFNVASDTVFLLDLIMNFRTGTVNEDSSEIILDPKVIKMNYLKSWFVVDFISSIPVDYIFLIVEKGMDSEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHNTYDLASAVVRIFNLIGMMLLLCHWDGCLQFLVPLLQDFPPDCWVSLNEMVNDSWGKQYSYALFKAMSHMLCIGYGAQAPVSMSDLWITMLSMIVGATCYAMFVGHATALIQSLDSSRRQYQEKYKQVEQYMSFHKLPADMRQKIHDYYEHRYQGKIFDEENILNELNDPLREEIVNFNCRKLVATMPLFANADPNFVTAMLSKLRFEVFQPGDYIIREGAVGKKMYFIQHGVAGVITKSSKEMKLTDGSYFGEICLLTKGRRTASVRADTYCRLYSLSVDNFNEVLEEYPMMRRAFETVAIDRLDRIGKKNSILLQKFQKDLNTGVFNNQENEILKQIVKHDREMVQAIAPINYPQMTTLNSTSSTTTPTSRMRTQSPPVYTATSLSHSNLHSPSPSTQTPQPSAILSPCSYTTAVCSPPVQSPLAARTFHYASPTASQLSLMQQQPQQQVQQSQPPQTQPQQPSPQPQTPGSSTPKNEVHKSTQALHNTNLTREVRPFSAWQPSLPHEVSILISRPHPTVGESLASIPQPVTAVPGTGLQAGGRSTVPQRVTFFRQMXSGAIPPNRGVLPAPLPLITPHPKK

[0332] DNA and Predicted Amino Acid Sequence of hBCNG-2

[0333] GenBank accession number AF064877. hBCNG-2 DNA sequenceGCGAGGAGGCGGGCCCGGCGGGGGAGCCGCGCGGCAGCCAGGCCACTTCATGCAGCGCCAGTTCGGCGCGCTCCTGCAGCCGGGCGTCAACAAGTTCTCGCTGCGGATGTTCGGCAGCCAGAAGGCCGTGGAGCGCGAGCAGGAGCGCGTCAAGTCGGCGGGGGCCTGGATCATCCACCCGTACAGCGACTTCAGGTTCTACTGGGACTTCACCATGCTGCTGTTCATGGTGGGAAACCTCATCATCATCCCAGTGGGCATCACCTTCTTCAAGGATGAGACCACTGCCCCGTGGATCGTGTTCAACGTGGTCTCGGACACCTTCTTCCTCATGGACCTGGTGTTGAACTTCCGCACCGGCATTGTGATCGAGGACAACACGGAGATCATCCTGGACCGCGAGAAGATCAAGAANAAGTATCTGCGCACGTGGTTCGTGGTGGTCTTCGTGTCCTCCATCCCCGTGGACTACATCTTCCTTATCGTGGAGAAGGGCATTGACTCCGAGGTCTACAAGACGGCACGCGCCCTGCGCATCGTGCGCTTCACCAAAATCCTCAGCCTCCTGCGGCTGCTGCGCCTCTCACGCCTGATCCGCTACATCCATCAGTGGGAGGAGATCTTCCACATGACCTATGACCTGGCCAGCGCGGTGATGAGGATCTGCAATCTCATCAGCATGATGCTGCTGCTCTGCCACTGGGACGGCTGCCTGCAGTTCCTGGTGCCTATGCTGCAGGACTTCCCGCGCAACTGCTGGGTGTCCATCAATGGCATGGTGAACCACTCGTGGAGTGAACTGTACTCCTTCGCACTCTTCAAGGCCATGAGCCACATGCTGTGCATCGGGTACGGCCGGCAGGCGCCCGAAAGCATGACGGACATCTGGCTGACCATGCTCAGCATGATTGTGGGTGCCACCTGCTACGCCATGTTCATCGGCCACGCCACTGCCCTCATCCAGTCGCTGGACTCCTCGCGGCGCCAGTACCAGGAGAAGTACAAGCAGGTGGAGCAGTACATGTCCTTCCACAAGCTGCCAGCTGACTTCCGCCAGAAGATCCACGACTACTATGAACACCGTTACCAGGGCAAGATGTTTGACGAGGACAGCATCCTGGGCGAGCTCAACGGGCCCCTGCGGGAGGAGATCGTCAACTTCAACTGCCGGAAGCTGGTGGCCTCCATGCCGCTGTTCGCCAACGCCGACCCCAACTTCGTCACGGCCATGCTGACCAAGCTCAAGTTCGAGGTCTTCCAGCCGGGTGACTACATCATCCGCGAAGGCACCATCGGGAAGAAGATGTACTTCATCCAGCACGGCGTGGTCAGCGTGCTCACTAAGGGCAACAAGGAGATGAAGCTGTCCGATGGCTCCTACTTCGGGGAGATCTGCCTGCTCACCCGGGGCCGCCGCACGGCGANCGTGCGGGCTGACACCTACTGCCGCCTCTATTCCCTGAGCGTGGACAACTTCAACGAAGTGCTGGAGGAGTACCCCATGATGCGGCGCGCTTTCGAGACGGTGGCCATCGACCGCCTGGACCGCATCGGCAAGAAGAATTCCATCCTCCTGCACAAGGTGCAGCATGACCTCAACTCGGGCGTATTCAACAACCAGGAGAACGCCATCATCCAGGAGATCGTCAAGTACGACCGCGAGATGGTGCAGCAGGCCGAGCTGGGTCAGCGCGTGGGCTTTTTCCCGCCGCCGCCGCCGCCGCCGCAGGTCACTTCGGCCATCGCCACGCTGCAGCAGGCGGCGGCCATGAGCTTCTGCCCGCAGGTGGChBCNG-2 predicted amino acid sequenceEEAGPAGEPRGSQASFMQRQFGALLQPGVNKFSLRMFGSQKAVEREQERVKSAGAWIIHPYSDFRFYWDFTMLLFMVGNLIIIPVGITFFKDETTAPWIVFNVVSDTFFLMDLVLNFRTGIVIEDNTEIILDPEKIKXKYLRTWFVVVFVSSIPVDYIFLIVEKGIDSEVYKTARALRIVRFTKILSLLRLLRLSRLIRYIHQWEEIFHMTYDLASAVMRICNLISMMLLLCHWDGCLQFLVPMLQDFPRNCWVSINGMVNHSWSELYSFALFKAMSHMLCIGYGRQAPESMTDIWLTMLSMIVGATCYAMFIGHATALIQSLDSSRRQYQEKYKQVEQYMSFHKLPADFRQKIHDYYEHRYQGKMFDEDSILGELNGPLREEIVNFNCRKLVASMPLFANADPNFVTAMLTKLKFEVFQPGDYIIREGTIGKKMYFIQHGVVSVLTKGNKEMKLSDGSYFGEICLLTRGRRTAXVRADTYCRLYSLSVDNFNEVLEEYPMMRRAFETVAIDRLDRIGKKNSILLHKVQHDLNSGVFNNQENAIIQEIVKYDREMVQQAELGQRVGFFPPPPPPPQVTSAIATLQQAAAMSFCPQVA

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What is claimed is:
 1. An isolated nucleic acid encoding a BCNG protein or a portion thereof.
 2. An isolated nucleic acid encoding a BCNG-related protein or a portion thereof.
 3. The nucleic acid of claim 1, wherein the BCNG protein is encoded by the sequence shown in mBCNG-1 (ATCC Accession No. ______) (Seq.ID.No.:1), mBCNG-2 (Seq.ID.No.:5), mBCNG-3 (Seq.ID.No.:9), mBCNG-4 (Seq.ID.No.:11), hBCNG-1 (ATCC Accession No. ______) (Seq.ID.No.:3) or hBCNG-2 (Seq.ID.No.:7).
 4. The nucleic acid of claim 1 or 2, wherein the nucleic acid is DNA or RNA.
 5. The nucleic acid of claim 1 or 2, wherein the nucleic acid is cDNA.
 6. The nucleic acid of claim 5, wherein the cDNA has the nucleotide sequence shown in SEQ. ID. No.: 1 for mBCNG-1 (ATCC Accession No. ______), SEQ. ID. No.: 3 for hBCNG-1, SEQ. ID. No.: 5 for mBCNG-2, SEQ. ID. No.: 7 for hBCNG-2, SEQ. ID. No.: 9 for mBCNG-3, or SEQ. ID. No.:11 for mBCNG-4.
 7. A vector comprising the nucleic acid of claim 1 or
 2. 8. The vector of claim 7, wherein the vector comprises viral or plasmid DNA.
 9. An expression vector comprising the nucleic acid of claim 1 or 2 and regulatory elements.
 10. A host vector system which comprises the expression vector of claim 9 in a suitable host.
 11. A vector, which comprises cDNA encoding mBCNG-1 (ATCC Accession No. ______).
 12. A vector, which comprises cDNA encoding hBCNG-1 (ATCC Accession No. ______).
 13. The host vector system of claim 10, wherein the suitable host is a bacterial cell, a eukaryotic cell, a mammalian cell or an insect cell.
 14. An isolated BCNG protein.
 15. An isolated BCNG-related protein.
 16. The protein of claim 14, wherein the BCNG protein has substantially the same amino acid sequence shown in Seq.ID.No.:2 for mBCNG-1 (FIG. 8A), Seq.ID.No.:6 for mBCNG-2. (FIG. 8A), Seq.ID.No.:10 for mBCNG-3 (FIG. 8A), Seq.ID.No.12 for mBCNG-4 (FIG. 8A), Seq.ID.No.:4 for hBCNG-1 (FIG. 8A), Seq.ID.No.:8 for hBCNG-2 (FIG. 8A) or a portion thereof.
 17. The protein of claim 15, wherein the BCNG-related protein has substantial homology to the amino acid sequence shown in Seq.ID.No.:2 mBCNG-1 (FIG. 8A), Seq.ID.No.:6 for mBCNG-2 (FIG. 8A), Seq.ID.No.:10 for mBCNG-3 (FIG. 8A), Seq.ID.No.12 for mBCNG-4 (FIG. 8A), Seq.ID.No.:4 for hBCNG-1 (FIG. 8A), Seq.ID.No.:8 for hBCNG-2 (FIG. 8A) or a portion thereof.
 18. A composition comprising a nucleic acid, encoding a BCNG protein or a BCNG-related protein or a portion thereof and a carrier.
 19. The composition of claim 18, wherein the nucleic acid comprises substantially the same coding sequence as the coding sequence shown in SEQ. ID. No.: 1 for mBCNG-1, SEQ. ID. No.: 0.3 for hBCNG-1, SEQ. ID. No.: 5 for mBCNG-2, SEQ. ID. No.: 7 for hBCNG-2, SEQ. ID. No.: 9 for mBCNG-3, SEQ. ID. No.:11 for mBCNG-4 or portion thereof.
 20. A composition comprising a BCNG protein or a BCNG-related protein or portion thereof and a carrier.
 21. The composition of claim 20, wherein the BCNG protein or BCNG-related protein comprises substantially the same amino acid sequence as the amino acid sequence shown in Seq.ID.No.:2 mBCNG-1 (FIG. 8A), Seq.ID.No.:6 for mBCNG-2 (FIG. 8A), Seq.ID.No.:10 for mBCNG-3 (FIG. 8A), Seq.ID.No.12 for mBCNG-4 (FIG. 8A), Seq.ID.No.:4 for hBCNG-1 (FIG. 8A), Seq.ID.No.:8 for hBCNG-2 (FIG. 8A) or a portion thereof.
 22. A nucleic acid probe capable of specifically hybridizing with a nucleic acid encoding a BCNG protein or BCNG-related protein.
 23. A nucleic acid probe capable of specifically hybridizing with the nucleic acid of claim
 1. 24. The nucleic acid probe of claim 22, wherein the probe is capable of specifically hybridizing with Seq.ID.No:13, Seq.ID.No:14, Seq.ID.No:15, Seq.ID.No:16, Seq.ID.No:17, Seq.ID.No:18, Seq.ID.No:19, Seq.ID.No:20, Seq.ID.No:21, Seq.ID.No:21, Seq.ID.No:22, Seq.ID.No:23, Seq.ID.No:24, Seq.ID.No:25, Seq.ID.No:26, Seq.ID.No:27, Seq.ID.No:28, Seq.ID.No:29, Seq.ID.No:30, Seq.ID.No:31, Seq.ID.No:32, Seq.ID.No:33, or Seq.ID.No:34.
 25. A method for identifying a nucleic acid in a sample which encodes a BCNG protein or a BCNG-related protein which comprises: (a) contacting the sample with a nucleic acid probe capable of specifically hybridizing with nucleic acid encoding a BCNG protein or a BCNG-related protein under conditions permissive to the formation of a complex between the nucleic acid probe and the nucleic acid encoding the BCNG protein or the BCNG-related protein in the sample; (b) determining the amount of complex formed in step (a); and (c) comparing the amount of complex determined in step (b) with the amount of complex formed using an arbitrary sequence, a greater amount of complex formed With the BCNG-specific probe indicating the presence of a nucleic acid encoding a BCNG protein or a BCNG-related protein in the sample.
 26. The method of claim 25, step (a) further comprising amplifying the nucleic acid molecule encoding the BCNG protein or the BCNG-related protein.
 27. The method of claim 26, wherein the amplification comprises contacting the nucleic acid molecule from the sample with at least one amplification primer capable of specifically hybridizing to mBCNG-1 (Seq.ID.No.:1), mBCNG-2. (Seq.ID.No.:5)” mBCNG-3 (Seq.ID.No.:9), mBCNG-4 (Seq.ID.No.:11), hBCNG-1 (Seq.ID.No.:3) or hBCNG-2 (Seq.ID.No.:7) under conditions suitable for polymerase chain reaction.
 28. The method of claim 26, wherein the amplified nucleic acid molecule encoding the BCNG protein or the BCNG-related protein is detected by size fractionation.
 29. The method of claim 25, further comprising isolating the complex by size fractionation.
 30. The method of claim 25, wherein the nucleic acid probe is labeled with a detectable marker.
 31. The method of claim 3d, wherein the detectable marker is a radiolabeled molecule, a fluorescent molecule, an enzyme, a ligand, or a magnetic bead.
 32. The method of claim 25, wherein the probe comprises Seq.ID.No:13, Seq.ID.No:14, Seq.ID.No:15, Seq.ID.No:16, Seq.ID.No:17, Seq.ID.No:18, Seq.ID.No:19, Seq.ID.No:20, Seq.ID.No:21, Seq.ID.No:21, Seq.ID.No:22, Seq.ID.No:23, Seq.ID.No:24, Seq.ID.No:25, Seq.ID.No:26, Seq.ID.No:27, Seq.ID.No:28, Seq.ID.No:29, Seq.ID.No:30, Seq.ID.No:31, Seq.ID.No:32, Seq.ID.No:33, or Seq.ID.No:34.
 33. The method of claim 25, wherein the nucleic acid probe is capable of specifically hybridizing to mBCNG-1 (Seq.ID.No.:1), mBCNG-2 (Seq.ID.No.:5), mBCNG-3 (Seq.ID.No.:9), mBCNG-4 (Seq.ID.No.:11), hBCNG-1 (Seq.ID.No.:3) or hBCNG-2 (Seq.ID.No.:7).
 34. An isolated nucleic acid, previously unknown, identified by the method of claim
 25. 35. A method for testing whether a compound affects the expression of a BCNG protein or a BCNG-related protein which comprises: (a) contacting a sample which expresses a BCNG protein or a BCNG-related protein with a compound; (b) determining the amount of expression of BCNG protein or BCNG-related protein in the sample; and (c) comparing the amount of BCNG protein or BCNG-related protein expression determined in step (b) with the amount determined in the absence of the compound.
 36. A method for identifying a compound capable of interacting with a BCNG protein or a BCNG-related protein which comprises: (a) contacting a sample which expresses a BCNG protein or a BCNG-related protein with a compound under conditions permissive to formation of a complex between the compound and the BCNG protein or the BCNG-related protein; (b) determining the amount of complex formed between the compound and the BCNG protein or the BCNG-related protein; (c) comparing the amount of complex formed in step (b) with the amount formed in the absence of the compound, a greater amount of complex formed in the presence of the compound indicating the presence of a compound capable of interacting with a BCNG protein or a BCNG-related protein.
 37. A method for identifing a compound capable of modulating BCNG protein or BCNG-related protein activity which comprises: (a) contacting a sample which expresses a BCNG protein or a BCNG-related protein with a compound; (b) determining the amount of activity of the BCNG protein or BCNG-related protein in the sample; and (c) comparing the amount of activity of the BCNG protein or the BCNG-related protein determined in step (b) with the amount determined in the absence of the compound, an increase or decrease in activity indicating the presence of a compound capable of modulating the activity of the BCNG protein or the BCNG-related protein.
 38. The method of claim 37, step (a) comprising first introducing the nucleic acid encoding a BCNG protein or a BCNG-related protein into an expression system and causing the expression system to express the nucleic acid under conditions whereby a BCNG protein or a BCNG-related protein is produced.
 39. The method of claim 37, wherein step (b) comprises measuring the channel electrical current or intracellular calcium level in the presence of the compound.
 40. The method of claim 38, wherein the expression system comprises a cultured host cell.
 41. A compound, previously unknown, identified by the method of claim 35, 36, or
 37. 42. The method of claim 25, 35, 36, or 37, wherein the BCNG protein or the BCNG-related protein comprises mBCNG-1 (Seq.ID.No.:2), mBCNG-2 (Seq.ID.No.:6), mBCNG-3 (Seq.ID.No.:10), mBCNG-4 (Seq.ID.No.:12), hBCNG-1 (Seq.ID.No.:4) or hBCNG-2 (Seq.ID.No.:8) or a portion thereof.
 43. The method of claim 25, 35, 36, or 37, wherein the sample comprises a cell, cell lysate or cell-free translation.
 44. The method of claim 43, wherein the cell is a cardiac cell, a kidney cell, a hepatic cell, an airway epithelial cell, a muscle cell, a neuronal cell, a glial cell, a microglial cell, an endothelial cell, a mononuclear cell, a tumor cell, a mammalian cell, an insect cell, or a Xenopus oocyte.
 45. A compound, previously unknown, identified by the method of claim 35, 36, or
 37. 46. The method of claim 35, 36 or 37, wherein the compound is a peptide, a peptidomimetic, a nucleic acid, a polymer, or a small molecule.
 47. The method of claim 35, 36 or 37, wherein the compound is bound to a solid support.
 48. The method of claim 25, 35, 36, or 37, wherein the BCNG protein or the BCNG-related protein is ion channel protein or an ion channel subunit protein.
 49. The method of claim 35, 36, or 37, wherein the compound is an agonist or antagonist of ion channel activity
 50. The method of claim 37 wherein the modulation is increased ion flow rate or decreased ion flow rate.
 51. The method of claim 37 wherein the modulation is increased ion permissivity or decreased ion permissivity
 52. A method of modulating BCNG or BCNG-related protein activity in a sample, comprising contacting the sample with the compound of claim
 41. 53. A method of treating a condition in a subject which comprises administering to the subject an amount of the compound of claim 41, effective to treat the condition.
 54. A pharmaceutical composition which comprises the compounds of claim 41 and a pharmaceutically acceptable carrier.
 55. The pharmaceutical composition of claim 54, wherein the carrier is a diluent, an aerosol, a topical carrier, an aqueous solution, a nonaqueous solution or a solid carrier.
 56. A method for treating a condition in a subject which comprises administering to the subject an amount of the pharmaceutical composition of claim 54, effective to treat the condition in the subject.
 57. The method of claim 53 or 56, wherein the condition is a neurological, renal, pulmonary, hepatic, or cardiovascular condition.
 58. The method of claim 57, wherein the condition is epilepsy, Alzheimer's Disease, Parkinson's Disease, long QT syndrome, sick sinus syndrome, age-related memory loss, cystic fibrosis, sudden death syndrome or a pacemaker rhythm dysfunction.
 59. The method of claim 53 or 56, wherein the subject is a human.
 60. An antibody which binds specifically to the protein of claim 14 or
 15. 61. A cell capable of producing the antibody of claim
 60. 62. A method of identifying the protein of claim 14 or in a sample comprising: a) contacting the sample with the antibody of claim 58 under conditions permissive to the formation of a complex between the antibody and the protein; b) determining the amount of complex formed; and c) comparing the amount of complex formed in step (b) with the amount of complex formed in the absence of the antibody, the presence of an increased amount of complex formed in the presence of the antibody indicating identification of the protein in the sample. 